Gene therapy approaches to supply apolipoprotein A-I agonists and their use to treat dyslipidemic disorders

ABSTRACT

The invention relates to genetic approaches to supply nucleotide sequences encoding modified forms of the native forms of apolipoprotein A-I (ApoA-I): mature ApoA-I, preproApoA-I and proApoA-I; including native ApoA-I modified to contain ApoA-I agonists, peptides which mimic the activity of ApoA-I; ApoA-I superagonists, peptides which exceed the activity of native ApoA-I; and modified native ApoA-I having one or more amphipathic helices replaced by the nucleotide sequences of one or more ApoA-I agonists; for the treatment of disorders associated with dyslipoproteinemia, including cardiovascular disease, atherosclerosis, restenosis, hyperlipidemia, and other disorders such as septic shock.

1. INTRODUCTION

The invention relates to gene therapy approaches to supply nucleotidesequences encoding modified forms of the native forms of apolipoproteinA-I (ApoA-I) i.e., mature ApoA-I, preproApoA-I and proApoA-I; ApoA-Ipeptides; ApoA-I agonists and superagonists, peptides which mimic orexceed the activity of native ApoA-I; and the native ApoA-I gene for thetreatment of disorders associated with dyslipoproteinemia, includingcardiovascular disease, atherosclerosis, restenosis, hyperlipidemia, andother disorders such as septic shock.

2. BACKGROUND OF THE INVENTION

Circulating cholesterol is carried by plasma lipoproteins—particles ofcomplex lipid and protein composition that transport lipids in theblood. Low density lipoproteins (LDL), and high density lipoproteins(HDL) are the major cholesterol carriers. LDL are believed to beresponsible for the delivery of cholesterol from the liver (where it issynthesized or obtained from dietary sources) to extrahepatic tissues inthe body. The term “reverse cholesterol transport” describes thetransport of cholesterol from extrahepatic tissues to the liver where itis catabolized and eliminated. It is believed that plasma HDL particlesplay a major role in the reverse transport process, acting as scavengersof tissue cholesterol.

The evidence linking elevated serum cholesterol to coronary heartdisease is overwhelming. For example, atherosclerosis is a slowlyprogressive disease characterized by the accumulation of cholesterolwithin the arterial wall. Compelling evidence supports the concept thatlipids deposited in atherosclerotic lesions are derived primarily fromplasma LDL; thus, LDLs have popularly become known as the “bad”cholesterol. In contrast, HDL serum levels correlate inversely withcoronary heart disease—indeed, high serum levels of HDL are regarded asa negative risk factor. It is hypothesized that high levels of plasmaHDL are not only protective against coronary artery disease, but mayactually induce regression of atherosclerotic plaques (e.g., see Badimonet al., 1992, Circulation 86 (Suppl. III):86-94). Thus, HDL havepopularly become known as the “good” cholesterol.

2.1. CHOLESTEROL TRANSPORT

The fat-transport system can be divided into two pathways: an exogenousone for cholesterol and triglycerides absorbed from the intestine, andan endogenous one for cholesterol and triglycerides entering thebloodstream from the liver and other non-hepatic tissue.

In the exogenous pathway, dietary fats are packaged into lipoproteinparticles called chylomicrons which enter the bloodstream and delivertheir triglycerides to adipose tissue (for storage) and to muscle (foroxidation to supply energy). The remnant of the chylomicron, containingcholesteryl esters, is removed from the circulation by a specificreceptor found only on liver cells. This cholesterol then becomesavailable again for cellular metabolism or for recycling to extrahepatictissues as plasma lipoproteins.

In the endogenous pathway, the liver secretes a large, very-low-densitylipoprotein particle (VLDL) into the bloodstream. The core of VLDLsconsists mostly of triglycerides synthesized in the liver, with asmaller amount of cholesteryl esters (either synthesized in the liver orrecycled from chylomicrons). Two predominant proteins are displayed onthe surface of VLDLs, apoprotein B-100 and apoprotein E. When a VLDLreaches the capillaries of adipose tissue or of muscle, itstriglycerides are extracted resulting in a new kind of particle,decreased in size and enriched in cholesteryl esters but retaining itstwo apoproteins, called intermediate-density lipoprotein (IDL).

In human beings, about half of the IDL particles are removed from thecirculation quickly (within two to six hours of their formation),because they bind tightly to liver cells which extract their cholesterolto make new VLDL and bile acids. The IDL particles which are not takenup by the liver remain in the circulation longer. In time, theapoprotein E dissociates from the circulating particles, converting themto LDL having apoprotein B-100 as their sole protein.

Primarily, the liver takes up and degrades most of the cholesterol tobile acids, which are the end products of cholesterol metabolism. Theuptake of cholesterol containing particles is mediated by LDL receptors,which are present in high concentrations on hepatocytes. The LDLreceptor binds both apoprotein E and apoprotein B-100, and isresponsible for binding and removing both IDLs and LDLs from thecirculation. However, the affinity of apoprotein E for the LDL receptoris greater than that of apoprotein B-100. As a result, the LDL particleshave a much longer circulating life span than IDL particles—LDLscirculate for an average of two and a half days before binding to theLDL receptors in the liver and other tissues. High serum levels of LDL(the “bad” cholesterol) are positively associated with coronary heartdisease. For example, in atherosclerosis, cholesterol derived fromcirculating LDLs accumulates in the walls of arteries leading to theformation of bulky plaques that inhibit the flow of blood until a cloteventually forms, obstructing the artery causing a heart attack orstroke.

Ultimately, the amount of intracellular cholesterol liberated from theLDLs controls cellular cholesterol metabolism. The accumulation ofcellular cholesterol derived from VLDLs and LDLs controls threeprocesses: first, it reduces the cell's ability to make its owncholesterol by turning off the synthesis of HMGCOA reductase—a keyenzyme in the cholesterol biosynthetic pathway. Second, the incomingLDL-derived cholesterol promotes storage of cholesterol by activatingACAT—the cellular enzyme which converts cholesterol into cholesterylesters that are deposited in storage droplets. Third, the accumulationof cholesterol within the cell drives a feedback mechanism that inhibitscellular synthesis of new LDL receptors. Cells, therefore, adjust theircomplement of LDL receptors so that enough cholesterol is brought in tomeet their metabolic needs, without overloading. (For a review, seeBrown & Goldstein, In, The Pharmacological Basis Of Therapeutics, 8thEd., Goodman & Gilman, Pergamon Press, NY, 1990, Ch. 36, pp. 874-896).

2.2. REVERSE CHOLESTEROL TRANSPORT

In sum, peripheral (non-hepatic) cells obtain their cholesterol from acombination of local synthesis and the uptake of preformed sterol fromVLDLs and LDLs. In contrast, reverse cholesterol transport (RCT) is thepathway by which peripheral cell cholesterol can be returned to theliver for recycling to extrahepatic tissues, or excretion into theintestine in bile. The RCT pathway represents the only means ofeliminating cholesterol from most extrahepatic tissues, and is crucialto maintenance of the structure and function of most cells in the body.

The RCT consists mainly of three steps: (a) cholesterol efflux, theinitial removal of cholesterol from various pools of peripheral cells;(b) cholesterol esterification by the action of lecithin:cholesterolacyltransferase (LCAT), preventing a re-entry of effluxed cholesterolinto cells; and (c) uptake/delivery of HDL cholesteryl ester to livercells. The RCT pathway is mediated by HDLS. HDL is a generic term forlipoprotein particles which are characterized by their high density. Themain lipidic constituents of HDL complexes are various phospholipids,cholesterol (ester) and triglycerides. The most prominent apolipoproteincomponents are A-I and A-II which determine the functionalcharacteristics of HDL; furthermore minor amounts of apolipoproteinsC-I, C-II, C-III, D, E, J, etc. have been observed. HDL can exist in awide variety of different sizes and different mixtures of theabove-mentioned constituents dependent on the status of remodelingduring the metabolic RCT cascade.

The key enzyme involved in the RCT pathway is LCAT. LCAT is producedmainly in the liver and circulates in plasma associated with the HDLfraction. Cholesteryl ester transfer protein (CETP) and another lipidtransfer protein, phospholipid transfer protein (PLTP) contribute tofurther remodeling the circulating HDL population. CETP can movecholesteryl esters made by LCAT to other lipoproteins, particularlyApoB-containing lipoproteins, such as VLDL. HDL triglycerides can becatabolized by the extracellular hepatic triglyceride lipase, andlipoprotein cholesterol is removed by the liver via several mechanisms.

Each HDL particle contains at least one copy (and usually two to fourcopies) of ApoA-I. ApoA-I is synthesized by the liver and smallintestine as preproapolipoprotein which is secreted as a proprotein thatis rapidly cleaved to generate a mature polypeptide having 243 aminoacid residues. ApoA-I consists mainly of 6 to 8 different 22 amino acidrepeats spaced by a linker moiety which is often proline, and in somecases consist of a stretch made up of several residues. ApoA-I formsthree types of stable structures with lipids: small, lipid-poorcomplexes referred to as pre-beta-1 HDL; flattened discoidal particlescontaining only polar lipids (phospholipid and cholesterol) referred toas pre-beta-2 HDL; and spherical particles containing both polar andnonpolar lipids, referred to as spherical or mature HDL (HDL₃ and HDL₂).Most HDL in the circulating population contain both ApoA-I and ApoA-II(the second major HDL protein) and are referred to herein as theAI/AII-HDL fraction of HDL. However, the fraction of HDL containing onlyApoA-I (referred to herein as the AI-HDL fraction) appear to be moreeffective in RCT. Certain epidemiologic studies support the hypothesisthat the AI-HDL fraction is antiartherogenic. (Parra et al., 1992,Arterioscler. Thromb. 12:701-707; Decossin et al., 1997, Eur. J. Clin.Invest. 27:299-307)

Although the mechanism for cholesterol transfer from the cell surface isunknown, it is believed that the lipid-poor complex, pre-beta-1 HDL isthe preferred acceptor for cholesterol transferred from peripheraltissue involved in RCT. Cholesterol newly transferred to pre-beta-1 HDLfrom the cell surface rapidly appears in the discoidal pre-beta-2 HDL.PLTP may increase the rate of disc formation, but data indicating a rolefor PLTP in RCT is lacking. LCAT reacts preferentially with discoidaland spherical HDL, transferring the 2-acyl group of lecithin or otherphospholipids to the free hydroxyl residue of fatty alcohols,particularly cholesterol to generate cholesteryl esters (retained in theHDL) and lysolecithin. The LCAT reaction requires ApoA-I as activator;i.e., ApoA-I is the natural cofactbr for LCAT. The conversion ofcholesterol to its ester sequestered in the HDL prevents re-entry ofcholesterol into the cell, the result being that cholesteryl esters aredestined for removal. Cholesteryl esters in the mature HDL particles inthe AI-HDL fraction (i.e., containing ApoA-I and no ApoA-II) are removedby the liver and processed into bile more effectively than those derivedfrom HDL containing both ApoA-I and ApoA-II (the AI/AII-HDL fraction).This may be due, in part, to the more effective binding of AI-HDL to thehepatocyte membrane. The existence of an HDL receptor has beenhypothesized, and recently a scavenger receptor, SR-BI, was identifiedas an HDL receptor. (Acton et al., 1996, Science 271:518-520). The SR-BIis expressed most abundantly in steroidogenic tissues (e.g., theadrenals), and in the liver. (Landshulz et al., 1996, J. Clin. Invest.98:984-995; Rigotti et al., 1996, J. Biol. Chem. 271:33545-33549).

CETP does not appear to play a major role in RCT, and instead isinvolved in the metabolism of VLDL-and LDL-derived lipids. However,changes in CETP activity or its acceptors, VLDL and LDL, play a role in“remodeling” the HDL population. For example, in the absence of CETP,the HDLs become enlarged particles which are not cleared. (For reviewson RCT and HDLs, see Fielding & Fielding, 1995, J. Lipid Res.36:211-228; Barrans et al., 1996, Biochem. Biophys. Acta. 1300:73-85;Hirano et al., 1997, Arterioscler. Thromb. Vasc. Biol. 17(b):1053-1059.

2.3. CURRENT TREATMENTS FOR LOWERING SERUM CHOLESTEROL

A number of treatments are currently available for lowering serumcholesterol and triglycerides (see, e.g., Brown & Goldstein, supra).However, each has its own drawbacks and limitations in terms ofefficacy, side-effects and qualifying patient population.

Bile-acid-binding resins are a class of drugs that interrupt therecycling of bile acids from the intestine to the liver; e.g.,cholestyramine (Questran Light®, Bristol-Myers Squibb), and colestipolhydrochloride (Colestid®, The Upjohn Company). When taken orally, thesepositively-charged resins bind to the negatively charged bile acids inthe intestine. Because the resins cannot be absorbed from the intestine,they are excreted carrying the bile acids with them. The use of suchresins, however, at best only lowers serum cholesterol levels by about20%, and is associated with gastrointestinal side-effects, includingconstipation and certain vitamin deficiencies. Moreover, since theresins bind other drugs, other oral medications must be taken at leastone hour before or four to six hours subsequent to ingestion of theresin; thus, complicating heart patient's drug regimens.

The statins are cholesterol lowering agents that block cholesterolsynthesis by inhibiting AMGCOA reductase—the key enzyme involved in thecholesterol biosynthetic pathway. The statins, e.g., lovastatin(Mevacor®, Merck & Co., Inc.) and pravastatin (Pravachol®, Bristol-MyersSquibb Co.) are sometimes used in combination with bile-acid-bindingresins. The statins significantly reduce serum cholesterol and LDL-serumlevels, and slow progression of coronary atherosclerosis. However, serumHDL cholesterol levels are only slightly increased. The mechanism of theLDL lowering effect may involve both reduction of VLDL concentration andinduction of cellular expression of LDL-receptor, leading to reducedproduction and/or increased catabolism of LDLS. Side effects, includingliver and kidney dysfunction are associated with the use of these drugs(Physicians Desk Reference, Medical Economics Co., Inc., Montvale, N.J.1997). Recently, the FDA has approved atorvasatatin (an HMGroA reductaseinhibitor developed by Parke-Davis) (Warner Lambert) for the treatmentof rare but urgent cases of familial hypercholesterolemia (1995, Scrip20 (19):10).

Niacin, or nicotinic acid, is a water soluble vitamin B-complex used asa dietary supplement and antihyperlipidemic agent. Niacin diminishesproduction of VLDL and is effective at lowering LDL. It is used incombination with bile-acid binding resins. Niacin can increase HDL whenused at adequate doses, however, its usefulness is limited by seriousside effects when used at such doses.

Fibrates are a class of lipid-lowering drugs used to treat various formsof hyperlipidemia, (i.e., elevated serum triglycerides) which may alsobe associated with hypercholesterolemia. Fibrates appear to reduce theVLDL fraction and modestly increase HDL—however the effects of thesedrugs on serum cholesterol is variable. In the United States, fibrateshave been approved for use as antilipidemic drugs but have not receivedapproval as hypercholerolemia agents. For example, clofibrate(Atromid-S®, Wyeth-Ayerest Laboratories) is an antilipidemic agent whichacts (via an unknown mechanism) to lower serum triglycerides by reducingthe VLDL fraction. Although serum cholesterol may be reduced in certainpatient subpopulations, the biochemical response to the drug isvariable, and is not always possible to predict which patients willobtain favorable results. Atromid-S® has not been shown to be effectivefor prevention of coronary heart disease. The chemically andpharmacologically related drug, gemfibrozil (Lopid®, Parke-Davis) is alipid regulating agent which moderately decreases serum triglyceridesand VLDL cholesterol, and increases HDL cholesterol—the HDL₂ and HDL₃subfractions as well as both ApoA-I and A-II (i.e., the AI/AII-HDLfraction). However, the lipid response is heterogeneous, especiallyamong different patient populations. Moreover, while prevention ofcoronary heart disease was observed in male patients between 40-55without history or symptoms of existing coronary heart disease, it isnot clear to what extent these findings can be extrapolated to otherpatient populations (e.g., women, older and younger males). Indeed, noefficacy was observed in patients with established coronary heartdisease. Serious side-effects are associated with the use of fibratesincluding toxicity such as malignancy, (especially gastrointestinalcancer), gallbladder disease and an increased incidence in non-coronarymortality. These drugs are not indicated for the treatment of patientswith high LDL or low HDL as their only lipid abnormality (Physicians'Desk Reference, 1997, Medical Economics Co., Inc., Montvale, N.J.)

Oral estrogen replacement therapy may be considered for moderatehypercholesterolemia in post-menopausal women. However, increases in HDLmay be accompanied with an increase in triglycerides. Estrogen treatmentis, of course, limited to a specific patent population (postmenopausalwomen) and is associated with serious side effects including inductionof malignant neoplasms, gall bladder disease, thromboembolic disease,hepatic adenoma, elevated blood pressure, glucose intolerance, andhypercalcemia.

Thus, there is a need to develop safer drugs that are efficacious inlowering serum cholesterol, increasing HDL serum levels, preventingcoronary heart disease, and/or treating existing disease.

2.4. ApoA-I AS A TARGET

None of the currently available drugs for lowering cholesterol safelyelevate HDL levels and stimulate RCT—most appear to operate on thecholesterol transport pathway, modulating dietary intake, recycling,synthesis of cholesterol, and the VLDL population.

While it is desirable to find drugs that stimulate cholesterol effluxand removal, several potential targets in the RCT exist—e.g., LCAT, HDLand its various components (ApoA-I, ApoA-II and phospholipids), LTP, andCETP—and it is not known which target would be most effective atachieving desirable lipoprotein profiles and protective effects.Perturbation of any single component in the RCT pathway ultimatelyaffects the composition of circulating lipoprotein populations, and theefficiency of RCT.

Several lines of evidence based on data obtained in vivo implicate theHDL and its major protein component, ApoA-I, in the prevention ofatherosclerotic lesions, and potentially, the regression ofplaques—making these attractive targets for therapeutic intervention.First, an inverse correlation exists between serum ApoA-I (HDL)concentration and atherogenesis in man (Gordon & Rifkind, 1989, N. Eng.J. Med. 321:1311-1316; Gordon et al., 1989, Circulation 79:8-15).Indeed, specific subpopulations of HDL have been associated with areduced risk for atherosclerosis in humans. (Miller, 1987, Amer. Heart113:589-597; Cheung et al., 1991, Lipid Res. 32:383-394).

Second, animal studies support the protective role of ApoA-I (HDL).Treatment of cholesterol fed rabbits with ApoA-I or HDL reduced thedevelopment and progression of plaque (fatty streaks) in cholesterol-fedrabbits. (Koizumi et al., 1988, J. Lipid Res. 29:1405-1415; Badimon etal., 1989, Lab. Invest. 60:455-461; Badimon et al., 1990, J. Clin.Invest. 85:1234-1241). However, the efficacy varied depending upon thesource of HDL (Beitz et al., 1992, Prostaglandins, Leukotrienes andEssential Fatty Acids 47:149-152; Mezdour et al., 1995, Atherosclerosis113:237-246).

Third, direct evidence for the role of ApoA-I was obtained fromexperiments involving transgenic animals. The expression of the humangene for ApoA-I transferred to mice genetically predisposed todiet-induced atherosclerosis protected against the development of aorticlesions (Rubin et al., 11991, Nature 353:265-267). The ApoA-I transgenewas also shown to suppress atherosclerosis in ApoE-deficient mice and inApo(a) transgenic mice (Paszty et al., 1994, J. Clin. Invest.94:899-903; Plump et al., 1994, Proc. Natl. Acad. Sci. USA 91:9607-9611,Lin et al., 1994, J. Lipid Res. 35:2263-2266). Similar results wereobserved in transgenic rabbits expressing human ApoA-I (Duverger, 1996,Circulation 94:713-717; Duverger et al., 1996, Arterioscler. Thromb.Vasc. Biol. 16:1424-1429; Emmanuel et al., 1997, Artheriosclerosis1035:144), and in transgenic rats where elevated levels of human ApoA-Iprotected against atherosclerosis and inhibited restenosis followingballoon angioplasty (Burkey et al., 1992, Circulation, Supplement I,86:I-472, Abstract No. 1876; Burkey et al., 1995, J. Lipid Res.36:1463-1473).

The AI-HDL appear to be more efficient at RCT than the AI/AII-HDLfraction. Studies with mice transgenic for human ApoA-I or ApoA-I andApoA-II (AI/AII) showed that the protein composition of HDLsignificantly affects its role—AI-HDL is more anti-atterogenic thanAI/AII-HDL (Schultz et al., 1993, Nature 365:762-764). Parallel studiesinvolving transgenic mice expressing the human LCAT gene demonstratethat moderate increases in LCAT activity significantly changelipoprotein cholesterol levels, and that LCAT has a significantpreference for HDL containing ApoA-I (Francone et al., 1995, J. Clinic.Invest. 96:1440-1448; Beard et al., 1997, Nature Medicine 3,7:744-749).While these data support a significant role for ApoA-I in activatingLCAT and stimulating RCT, additional studies demonstrate a morecomplicated scenario: a major component of HDL that modulates efflux ofcell cholesterol is the phospholipid (Fournier et al., 1996, J. LipidRes. 37:1704-1711).

In view of the potential role of HDL, i.e., both ApoA-I and itsassociated phospholipid, in the protection against atheroscleroticdisease, human clinical trials utilizing recombinantly produced ApoA-Iwere commenced, discontinued and apparently recommenced by UCB Belgium(Pharma Projects, Oct. 27, 1995; IMS R&D Focus, Jun. 30, 1997; DrugStatus Update, 1997, Atherosclerosis 2(6):261-265); see also M. Erikssonat Congress, “The Role of HDL in Disease Prevention,” Nov. 7-9, 1996,Fort Worth; Lacko & Miller, 1997, J. Lipid Res. 38:1267-1273; andWO94/13819) and were commenced and discontinued by Bio-Tech(Pharmaprojects, Apr. 7, 1989). Trials were also attempted using ApoA-Ito treat septic shock (Opal, “Reconstituted HDL as a Treatment Strategyfor Sepsis,” IBCs 7th Annual International Conference on Sepsis, Apr.28-30, 1997, Washington, D.C.; Gouni et al., 1993, J. Lipid Res.94:139-146; Levine WO96/04916). However, there are many pitfallsassociated with the production and use of ApoA-I making it less thanideal as a drug; e.g., ApoA-I is a large protein that is difficult andexpensive to produce; significant manufacturing and reproducibilityproblems must be overcome with respect to stability during storage,delivery of an active product and half-life in vivo.

In view of these drawbacks, attempts have been made to prepare peptidesthat mimic ApoA-I. Since the key activities of ApoA-I have beenattributed to the presence of multiple repeats of a unique secondarystructural feature in the protein—a class A amphipathic α-helix(Segrest, 1974, FEBS Lett. 38:247-253), most efforts to design peptideswhich mimic the activity of ApoA-I have focused on designing peptideswhich form class A-type amphipathic α-helices.

Class A-type amphipathic α-helices are unique in that positively chargedamino acid residues are clustered at the hydrophobic-hydrophilicinterface and negatively charged amino acid residues are clustered atthe center of the hydrophilic face. Furthermore, Class A β-helicalpeptides have a hydrophobic angle of less than 180° (Segrest et al.,PROTEINS, 1990; Structure, Function and Genetics 8:103-117). The initialde novo strategies to design ApoA-I mimics were not based upon theprimary sequences of naturally occurring apolipoproteins, but ratherupon incorporating these unique Class A helix features into thesequences of the peptide analogues as well as some of the properties ofthe ApoA-I domains (see, e.g., Davidson et al., 1996, Proc. Natl. Acad.Sci. USA 93:13605-13610; Rogers et al., 1997, Biochemistry 36:288-300;Lins et al., 1993, Biochim. Biophys. Acta biomembranes 1151:137-142; Jiand Jonas, 1995, J. Biol. Chem. 270:11290-11297; Collet et al., 1997,Journal of Lipid Research, 38:634-644; Sparrow and Gotto, 1980, Ann.N.Y. Acad. Sci. 348:187-211; Sparrow and Gotto, 1982, CRC Crit. Rev.Biochem. 13:87-107; Sorci-Thomas et al., 1993, J. Biol. Chem.268:21403-21409; Wang et al., 1996, Biochim. Biophys. Acta 174-184;Minnich et al., 1992, J. Biol. Chem. 267:16553-16560; Holvoet et al.,1995, Biochemistry 34:13334-13342; Sorci-Thomas et al., 1997, J. Biol.Chem. 272 (11):7278-7284; and Frank et al., 1997, Biochemistry36:1798-1806).

In one study, Fukushima et al. synthesized a 22-residue peptide composedentirely of Glu, Lys and Leu residues arranged periodically so as toform an amphipathic α-helix with equal hydrophilic and hydrophobic faces(“ELK peptide”) (Fukushima et al., 1980, J. Biol. Chem..255:10651-10657; Fukushima et al., 1979, J. Amer. Chem. Soc.101(13):3703-3704). The ELK peptide shares 41% sequence homology withthe 198-219 fragment of ApoA-I. As studied by quantitativeultrafiltration, gel permeation chromatography and circular dichroism,this ELK peptide was shown to effectively associate with phospholipidsand mimic some of the physical and chemical properties of ApoA-I (Kaiseret al., 1983, Proc. Natl. Acad. Sci. USA 80:1137-1140; Kaiser et al.,1984, Science 223:249-255; Fukushima et al., 1980, supra; Nakagawa etal., 1985, J. Am. Chem. Soc. 107:7087-7092). Yokoyama et al. concludedfrom such studies that the crucial factor for LCAT activation is simplythe presence of a large enough amphipathic structure (Yokoyama et al.,1980, J. Biol. Chem. 255(15):7333-7339). A dimer of this 22-residuepeptide was later found to more closely mimic ApoA-I than the monomer;based on these results, it was suggested that the 44-mer which ispunctuated in the middle by a helix breaker (either Gly or Pro),represented the minimal functional domain in ApoA-I (Nakagawa et al.,1985, supra).

Another study involved model amphipathic peptides called “LAP peptides”(Pownall et al., 1980, Proc. Natl. Acad. Sci. USA 77(b):3154-3158;Sparrow et al., 1981, In: Peptides:Synthesis-Structure-Function, Rochand Gross, Eds., Pierce Chem. Co., Rockford, Ill., 253-256). Based onlipid binding studies with fragments of native apolipoproteins, severalLAP peptides were designed, designated LAP-16, LAP-20 and LAP-24(containing 16, 20 and 24 amino acid residues, respectively). Thesemodel amphipathic peptides share no sequence homology with theapolipoproteins and were designed to have polar faces organized in amanner unlike the class A-type amphipathic helical domains associatedwith apolipoproteins. From these studies, the authors concluded that aminimal length of 20 residues is necessary to confer lipid-bindingproperties to model amphipathic peptides.

Studies with mutants of LAP20 containing a proline residue at differentpositions in the sequence indicated that a direct relationship existsbetween lipid binding and LCAT activation, but that the helicalpotential of a peptide alone does not lead to LCAT activation (Ponsin etal., 1986 J. Biol. Chem. 261(20):9202-9205). Moreover, the presence ofthis helix breaker (Pro) close to the middle of the peptide reduced itsaffinity for phospholipid surfaces as well as its ability to activateLCAT. While certain of the LAP peptides were shown to bind phospholipids(Sparrow et al., supra), controversy exists as to the extent to whichLAP peptides are helical in the presence of lipids (Buchko et al., 1996,J. Biol. Chem. 271(6):3039-3045; Zhong et al., 1994, Peptide Research7(2):99-106).

Segrest et al. have synthesized peptides composed of 18 to 24 amino acidresidues that share no sequence homology with the helices of ApoA-I(Kannelis et al., 1980, J. Biol. Chem. 255(3):11464-11472; Segrest etal., 1983, J. Biol. Chem. 258:2290-2295). The sequences werespecifically designed to mimic the amphipathic helical domains of classA exchangeable apolipoproteins in terms of hydrophobic moment (Eisenberget al., 1982, Nature 299:371-374) and charge distribution (Segrest etal., 1990, Proteins 8:103-117; U.S. Pat. No. 4,643,988). One 18-residuepeptide, the “18A” peptide, was designed to be a model class-A α-helix(Segrest et al., 1990, supra). Studies with these peptides and otherpeptides having a reversed charged distribution, like the “18R” peptide,have consistently shown that charge distribution is critical foractivity; peptides with a reversed charge distribution exhibit decreasedlipid affinity relative to the 18A class-A mimics and a lower helicalcontent in the presence of lipids (Kanellis et al., 1980, J. Biol. Chem.255:11464-11472; Anantharamaiah et al., 1985, J. Biol. Chem.260:10248-10255; Chung et al., 1985, J. Biol. Chem. 260:10256-10262;Epand et al., 1987, J. Biol. Chem. 262:9389-9396; Anantharamaiah et al.,1991, Adv. Exp. Med. Biol. 285:131-140).

Other synthetic peptides sharing no sequence homology with theapolipoproteins which have been proposed with limited success includedimers and trimers. of the 18A peptide (Anatharamaiah et al., 1986,Proteins of Biological Fluids 34:63-66), GALA and EALA peptides(Subbarao et al., 1988, Proteins: Structure, Function and Genetics3:187-198) and ID peptides (Labeur et al., 1997, Arteriosclerosis,Thrombosis, and Vascular Biology 17:580-588) and the 18AM4 peptide(Brasseur et al., 1993, Biochem. Biophys. Acta 1170:1-7).

A “consensus” peptide containing 22-amino acid residues based on thesequences of human ApoA-I has also been designed (Venkatachalapathi etal., 1991, Mol. Conformation and Bio. Interactions B:585-596). Thesequence was constructed by identification of the most prevalent residueat each position of the helices of ApoA-I. Like the peptides describedabove, the helix formed by this peptide has positively charged aminoacid residues clustered at the hydrophilic:hydrophobic interface,negatively charged amino acid residues clustered at the center of thehydrophilic face and hydrophobic angle of less than 180°. While a dimerof this peptide is somewhat effective in activating LCAT, the monomerexhibited poor lipid binding properties (Vekatachalapathi et al., 1991,supra).

Based primarily on in vitro studies with the peptides described above, aset of “rules” has emerged for designing peptides which mimic thefunction of ApoA-I. significantly, it is thought that an amphipathicα-helix having positively charged residues clustered at thehydrophilic-hydrophobic interface and negatively charged amino acidresidues clustered at the center of the polar face is required for lipidaffinity and LCAT activation (Venkatachalapathi et al., 1991, supra).Anantharamaiah et al., have also indicated that the negatively chargedGlu residue at position 13 of the consensus 22-mer peptide, which ispositioned within the hydrophobic face of the α-helix, plays animportant role in LCAT activation (Anantharamaiah et al., 1991, supra).Furthermore, Brasseur has indicated that a hydrophobic angle (pho angle)of less than 180° is required for optimal lipid-apolipoprotein complexstability, and also accounts for the formation of discoidal particleshaving the peptides around the edge of a lipid bilayer (Brasseur, 1991,J. Biol. Chem. 66(24):16120-16127). Rosseneu et al., have also insistedthat a hydrophobic angle of less than 180° is required for LCATactivation (WO93/25581).

However, despite these “rules” to date, no one has designed or produceda peptide as active as ApoA-I—the best having less than 40% of theactivity of ApoA-I as measured by the LCAT activation assay describedherein. None of the peptide “mimetics” described in the literature havenot been demonstrated to be useful as a drug.

In view of the foregoing, there is a need for the development of astable ApoA-I agonist that mimics the activity of ApoA-I, which isrelatively simple and cost-effective to produce. However, the “rules”for designing efficacious ApoA-I mimetics have not been unraveled andthe principles to devise organic molecules with the function of ApoA-Iare unknown.

3. SUMMARY OF THE INVENTION

The invention relates to genetic approaches to supply nucleotidesequences encoding modified forms of the native forms of apolipoproteinA-I (ApoA-I): mature ApoA-I, preproApoA-I and proApoA-I; includingnative. ApoA-I modified to contain ApoA-I agonists, peptides which mimicthe activity of ApoA-I; ApoA-I superagonists, peptides which exceed theactivity of native ApoA-I; and modified native ApoA-I having one or moreamphipathic helices replaced by the nucleotide sequences of one or moreApoA-I agonists; for the treatment of disorders associated withdyslipoproteinemia, including cardiovascular disease, atherosclerosis,restenosis, hyperlipidemia, and other disorders such as septic shock.

In particular, the invention relates to genetic approaches. to supplynucleotide sequences encoding modified native forms of the ApoA-Iprotein and peptides which act as ApoA-I agonists capable of formingamphipathic α-helices (in the presence of lipids) that approach orsurpass the activity of native ApoA-I, e.g., formation of pre-β-like orHDL-like complexes, promotion of cholesterol efflux, binding to lipids,increasing the activity of LCAT, and increasing serum levels of HDL.

The present invention also relates to nucleotide sequences encoding amodified native ApoA-I having one or more amphipathic helices replacedby the nucleotide sequences of one or more ApoA-I agonists.

The present invention relates to DNA vectors or cassettes that containnucleotide sequences encoding modified ApoA-I or peptides which act asApoA-I agonists or superagonists. The invention further relates to DNAvectors or cassettes that contain nucleotide sequences encoding modifiedforms of the native ApoA-I gene. The DNA vectors or cassettes may encodemodified forms of native ApoA-I or ApoA-I agonists under the control ofstrong regulatory elements which result in high levels of ApoA-Iexpression. To increase efficiency of production, the DNA expressionvectors or cassettes may be designed to encode multiple units of thepeptide, e.g., the cassettes may contain nucleotide sequences encodingseveral ApoA-I peptides in tandem, separated by an internal ribosomebinding site, so that several peptides may be encoded by the samecassette. The vectors or cassettes may also express ApoA-I peptides asmonopolymers (repeating peptide units) or heteropolymers (differentpeptide units). The cassettes of the present invention may alsoadditionally encode prepropeptide or propeptide ApoA-I sequences so thatthe peptides of the present invention are processed in the same way asthe native ApoA-I protein.

The present invention further relates to genetically engineered hostcells which express the modified native forms of ApoA-I: mature ApoA-I,preproform or proform, peptides which mimic ApoA-I activity or thenative ApoA-I gene product. The host cells may be genetically engineeredin vitro or in vivo. The present invention also relates to transgenicanimals engineered to express the ApoA-I peptides of the presentinvention.

The invention also relates to in vivo delivery applications for genetherapy, including administering engineered viral vectors or liposomeswhich contain DNA sequences encoding ApoA-I or ApoA-I agonist peptidesin monomeric or heteromeric forms. The invention relates to ex vivo genetherapy approaches to provide engineered cells derived from a patient,or a compatible host, to express modified forms of the native ApoA-Igene,-ApoA-I agonists in monomeric or heteromeric form. Ex vivo genetherapy approaches may also encompass delivery of “sacs of cells” whichhave been genetically engineered to express the ApoA-I gene of thepresent invention, including, ApoA-I agonists in monomeric orheteromeric form and additional ApoA-I prosequences. In a preferredembodiment, human hepatocytes may be engineered to express ApoA-Iagonists.

The invention is based, in part, on the Applicants' design and discoveryof peptides that mimic the helical structure and amphipathic propertiesof the helical amphipathic domains of ApoA-I. Surprisingly, the peptidesof the invention have a ApoA-I activity, i.e., formation of HDL-likecomplexes and promotion of cholesterol efflux, well above those reportedfor ApoA-I-derived peptides described in the literature. Indeed, someembodiments of the invention approach 100% of the activity of nativeApoA-I, whereas superagonists described herein exceed the specificactivity of ApoA-I.

The invention is illustrated by way of working examples that describethe in vitro and in vivo activity and efficacy of the ApoA-I peptidesand agonists encoded by the nucleic acids of the present invention.Based upon the structure and activity of the exemplified embodiments,the Applicants have devised a set of “rules” which can be used to designaltered or mutated forms that are also within the scope of theinvention.

The invention also relates to pharmaceutical formulations containingsuch cell lines and vectors encoding modified forms of the nativeApoA-I, i.e., mature ApoA-I, preproform of ApoA-I or proform of ApoA-I,or ApoA-I agonists (either as peptides or peptide-lipid complexes) asthe active ingredient, as well as methods for preparing suchformulations and their use to treat diseases associated withdyslipoproteinemia (e.g., cardiovascular diseases, atherosclerosis),restenosis, hyperlipidemia, hypertriglyceridemia, and/or endotoxinemia(e.g., septic shock).

3.1. ABBREVIATIONS

As used herein, the abbreviations for the genetically encoded aminoacids are as follows: One-Letter Common Amino Acid Symbol AbbreviationAlanine A Ala Arginine R Arg Asparagine N Asn Aspartic acid D AspCysteine C Cys Glutamine Q Gln Glutamic acid E Glu Glycine G GlyHistidine H His Isoleucine I Ile Leucine L Leu Lysine K Lys Methionine MMet Phenylalanine F Phe Proline P Pro Serine S Ser Threonine T ThrTryptophan W Trp Tyrosine Y Tyr Valine V Val

3.2. DEFINITIONS

As used herein, the following terms shall have the following meanings:

“Hydrophophilic Amino Acid” refers to an amino acid having a side chainthat tends to associate with the aqueous phase or polar head of aphospholipid when in the presence of lipids. Genetically encodedhydrophilic amino acids include Asp (D), Glu (E), His (H), Lys (K), Arg(R), Asn (N), Gln (Q), Ser (S) and Thr (T).

“Acidic Amino Acid” refers to an amino acid having a side chain pK valueof less than 7. Acidic amino acids typically have negatively chargedside chains at physiological pH due to loss of a hydrogen ion.Genetically encoded acidic amino acids include Glu (E) and Asp (D).

“Basic Amino Acid” refers to an amino acid having a side chain pK valueof greater than 7. Basic amino acids typically have positively chargedside chains at physiological pH due to association with hydronium ion.Genetically encoded basic amino acids include Arg (R) and Lys (K).

“Polar Amino Acid” refers to an amino acid having a side chain that isuncharged at physiological pH, but which has at least one bond whereinthe pair of electrons shared in common by two atoms is held more closelyby one of the atoms. Genetically encoded polar amino acids include Asn(N) and Gln (Q).

“Hydrophobic Amino Acid” refers to an amino acid having a side chainthat tends to associate with the acyl chains of a phospholipid when inthe presence of lipids. Genetically encoded hydrophobic amino acidsinclude Phe (F), Tyr (Y), Trp (W), Leu (L), Val (V), Ile (I), Met (M),Gly (G), Ala (A) and Pro (P).

“Aromatic Amino Acid” refers to a hydrophobic amino acid with a sidechain having at least one aromatic or heteroaromatic ring. The aromaticor heteroaromatic ring may contain one or more substituents. Geneticallyencoded aromatic amino acids include Phe (F), Tyr (Y) and Trp (W).

“Nonpolar Amino Acid” refers to a hydrophobic amino acid having a sidechain that is uncharged at physiological pH and which has bonds whereinthe pair of electrons shared in common by two atoms is generally heldequally by each at the two atoms (i.e., the side chain is not polar).

“AliPhatic Amino Acid” refers to a hydrophobic amino acid having analiphatic hydrocarbon side chain. Genetically encoded aliphatic aminoacids include Ala (A), Val (V), Leu (L) and le (I).

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The nucleotide sequence (SEQ. ID NO.: ______) and amino acidsequence (SEQ. ID NO.: ______) for human ApoAI.

FIG. 2. Nucleotide sequences which encode core peptides of structure I(SEQ. ID NOS.: ______).

FIG. 3. Nucleotide sequences which encode core peptides of structure II(SEQ. ID NOS.: ______).

FIG. 4. Nucleotide sequences which encode core peptides of structure III(SEQ. ID NOS.: ______).

5. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to gene therapy approaches to providenucleotide sequences encoding modified forms of the native ApoA-I gene,nucleotide sequences encoding ApoA-I agonists which mimic ApoA-Ifunction and activity or surpass native ApoA-I function and activity,for the treatments of disorders associated with hypercholesterolemia.

The nucleotide sequences of the present invention encode peptides whichform amphipathic helices (in the presence of lipids), bind lipids, formpre-β like or HDL-like complexes, activate LCAT, increase serum HDLconcentration and promote cholesterol efflux.

The biological function of encoded peptides appears to correlate withtheir helical structures, or conversion to helical structures in thepresence of lipids.

At least three types of DNA vectors or cassettes can be designed andconstructed in accordance with the present invention, to containnucleotide sequences of the native human ApoA-I gene; modified forms ofthe native or proform of ApoA-I; nucleotide sequences encoding ApoA-Iagonists in preproform and proform; and nucleotide sequences encodingApoA-I agonists and superagonists. The DNA vectors or cassettes may alsoexpress the ApoA-I peptides as homopolymers (repeating peptide units) orheteropolymers (different peptide units) in order to increase efficiencyof production.

The invention further relates to genetically engineered host cells whichexpress the ApoA-I peptide agonists of the present invention. The hostcells may be engineered in vivo or in vitro. The present inventionfurther relates to in vivo and in vitro gene therapy approaches toexpress ApoA-I peptide agonists in a host. The present invention alsorelates to transgenic animals engineered to express the modified formsof the native or proform of ApoA-I and ApoA-I peptide agonists of thepresent invention.

The nucleotide sequences encoding ApoA-I agonists of the invention canbe provided as naked RNA or DNA sequences, in vectors, such as viralvectors or by transfected cells. The invention includes thepharmaceutical formulations and the use of such preparations in thetreatment of dyslipoproteinemia, hyperlipidemia, hypercholesterolemia,coronary heart disease, atherosclerosis, and other conditions such asendotoxinemia/septic shock.

The invention is based, in part, on the Applicant's discovery that theApoA-I agonists of the invention associate with the HDL component ofplasma, and can increase the concentration of HDL and pre-β particles.The ApoA-I agonists of the invention increase cholesterol efflux fromextrahepatic tissues. The agonists are also extremely efficient atactivating LCAT, and thus promote RCT. Use of the ApoA-I agonists of theinvention in vivo in animal models results in an increase in serum HDLconcentration.

The invention is set forth in more detail in the subsections below,which describe: the nucleotide sequences encoding the native form ofApoA-I, the preproform of ApoA-I, modified forms of the native orproform of ApoA-I and ApoA-I peptide agonists, the vectors and celllines which may be designed to express these nucleotide sequences; invivo and ex vivo gene therapy approaches to provide these nucleotidesequences; and methods of preparation of bulk and unit dosageformulations; and methods of use.

5.1. NUCLEOTIDE SEQUENCES ENCODING ApoA-I AND ApoA-I AGONISTS

The nucleotide sequences of the present invention encode ApoA-I peptidesor ApoA-I agonists which are capable of forming regions of amphipathicα-helices in the presence of lipids and which mimic the activity ofApoA-I. Nucleotide sequences encoding the ApoA-I agonists and ApoA-Ipeptides are described herein.

The present invention also encompasses nucleotide sequences encoding thefull length ApoA-I gene (SEQ. ID NO.: ______ (FIG. 1)) which have beenmodified to enhance or increase the ApoA-I like activities of theexpressed gene product, i.e., form amphipathic helices (in the presenceof lipids), bind lipids, form pre-β-like or HDL-like complexes, activateLCAT, increase serum HDL concentration and promote cholesterol efflux.The present invention also encompasses nucleotide sequences which encodemodified forms of native ApoA-I, i.e., nucleotide sequences encoding amodified ApoA-I having one, two or more amphipathic helices replaced byat least two Apo-I agonists.

The present invention also relates to nucleotide sequences which encodemodified forms of ApoA-II, ApoA-III, ApoA-IV, ApoB, ApoC-I, ApoC-II,ApoC-III, ApoD and ApoE which have been modified to encode one, two ormore ApoA-I agonists of the present invention (see e.g., Lusis, 1988, Jof Lipid Res. 29:397-428, incorporated herein by reference in itsentirety).

The nucleotide sequences of the present invention also encompassnucleotide sequences encoding core peptides of Structure I (e.g., SEQ IDNO.: ______ (FIG. 2)), core peptides of Structure II (e.g., SEQ ID NO.:______ (FIG. 3)) or core peptides of Structure III (e.g., SEQ ID NO.:______ (FIG. 4)).

The invention also encompasses:

-   -   (a) DNA vectors that contain any of the foregoing ApoA-I coding        sequences and/or their complements (i.e., antisense);    -   (b) DNA expression vectors that contain any of the foregoing        ApoA-I coding sequences operatively associated with a regulatory        element that directs the expression of the coding sequences;    -   (c) DNA expression vectors or cassettes encoding ApoA-I agonists        in tandem separated by a internal ribosome binding site so that        several of the same coding sequence may be expressed from the        same cassette or several different coding sequences expressing        different ApoA-I agonists may be expressed from the same        cassette;    -   (d) genetically engineered host cells that contain any of the        foregoing ApoA-I coding sequences operatively associated with a        regulatory element that directs the expression of the coding        sequences in the host cell.

As used herein, regulatory elements include, but are not limited toinducible and non-inducible promoters, enhancers, operators and otherelements known to those skilled in the art that drive and regulateexpression. Such regulatory elements include but are not limited to thecytomegalovirus hCMV immediate early gene, the early or late promotersof the SV40 adenovirus, the lac system, the trp system, the TAC system,the TRC system, the major operator and promoter regions of phage A, thecontrol regions of the fd coat protein, the promoter for3-phosphoglycerate kinase, the promoters of acid phosphatase, and thepromoters of the yeast α-mating factors.

5.1.1. THE NATIVE APoA-I GENE

The ApoA-I gene encodes the principal protein constituent of HDL and isassociated with the regulation of cholesterol efflux and the serum HDLconcentration. Nucleic acids encoding the ApoA-I gene of the presentinvention are described herein for use in gene therapy approaches fortreating disorders associated with dyslipidemia. As used herein theApoA-I gene of the present invention includes, but is not limited to:

-   -   (a) a nucleic acid molecule containing the cDNA encoding human        ApoA-I, the full length human ApoA-I cDNA comprises 842        nucleotides (SEQ ID NO.:______ (FIG. 1)) (see Shoulders et al.,        1983, Nucleic Acids Research 11:2827-2837, incorporated herein        by reference in its entirety) in which nucleotide sequences        encoding at least one of the 22 amino acid repeats are replaced        by nucleotide sequences encoding the ApoA-I agonists of the        present invention;    -   (b) a nucleic acid molecule containing the genomic sequence of        human ApoA-I which contains at least two introns of 185 and 588        nucleotides (see Shoulders et al., 1983, Nucleic Acids Research        11:2827-2837, incorporated herein by reference in its entirety);    -   (c) a nucleic acid molecule containing the DNA sequences        encoding 1 to 8 of the different 22 amino acid repeats found in        the native human ApoA-I, and may further contain the DNA        sequences encoding the linker or spacer moieties found between        one or more of the repeats;    -   (d) a nucleic acid molecule containing the DNA sequences        encoding a modified ApoA-I in which at least one amphipathic        helix is replaced by the sequence of at least one ApoA-I        agonist;    -   (e) a nucleic acid molecule containing all or a portion of (a),        (b), (c) or (d) and additionally the DNA sequences encoding the        prepropeptide segment or leader peptide which encompasses the        first 18 amino acids of the amino terminus of ApoA-I. The leader        peptide of human ApoA-I, 5′MKAAVLTLAVLFLTGSQA3′ (SEQ. ID NO.:        ______), is cleaved following secretion of ApoA-I from the host        cell;    -   (f) a nucleic acid molecule containing all or a portion of (a),        (b), (c), (d) or (e) and additionally the DNA sequences encoding        the propeptide segment which encompasses a 6 amino acid        sequence, 5′RHFWQQ3′, which is cleaved by specific proteases        resulting in a mature ApoA-I; and/or    -   (g) a nucleic acid molecule containing a DNA sequence encoding a        gene product functionally equivalent to an ApoA-I peptide.

The term “functionally equivalent to an ApoA-I peptide” as used herein,refers to a gene product that exhibits one of the biological activitiesof an ApoA-I peptide including formation of amphipathic helices (in thepresence of lipids), binding to lipids, formation of pre-β-like orHDL-like complexes, activation of LCAT, increasing serum HDLconcentration and promotion of cholesterol efflux. According to thisaspect, the invention also includes nucleotide sequences which encodemodified forms of ApoA-II, ApoA-III, ApoA-IV, ApoB, ApoC-I, ApoC-II,ApoC-III, ApoD and ApoE which have been modified to encode one, two ormore ApoA-I agonists of the present invention.

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, theDNA sequences described above encoding native ApoA-I. Such hybridizationconditions may be highly stringent or less highly stringent, asdescribed above. In instances wherein the nucleic acid molecules aredeoxyoligonucleotides (“oligos”), highly stringent conditions may refer,e.g., to washing in 6×SSC/0.05% sodium pyrophosphate at 37° C. (for14-base oligos), 48° C. (for 17-base oligos), 55° C. (for 20-baseoligos), and 60° C. (for 23-base oligos). These nucleic acid moleculesmay act as ApoA-I agonist antisense molecules, useful, for example, inApoA-I gene regulation, for and/or as antisense primers in amplificationreactions of ApoA-I nucleic acid sequences. With respect to ApoA-I generegulation, such techniques may be used to regulate, for example ahypercholesterolemia disorder. Further, such sequences may be used aspart of ribozyme and/or triple helix sequences, also useful for generegulation of mutant forms of ApoA-I which may display ApoA-I antagonistproperties. Still further, such molecules may be used as components ofdiagnostic methods whereby, for example, the presence of a particularmutant ApoA-I allele responsible for causing a hypercholesterolemiadisorder may be detected.

In addition to those human sequences described herein, additional ApoA-Igene sequences can. be identified and readily isolated, with out undueexperimentation, by molecular biological techniques well known in theart.

5.1.2. ApoA-I PEPTIDES AND ApoA-I AGONISTS

The nucleotide sequences may encode ApoA-I peptides or agonists whichhave as their main feature a “core” peptide. composed of 15 to 23 aminoacid residues. The amino acid sequence of this core peptide is basedupon the amino acid sequences of a family of peptides which theApplicants have discovered mimic the structure and amphipathic helicalproperties of ApoA-I, and which exhibit specific activities thatapproach, or in some embodiments even exceed, the specific activity ofnative ApoA-I.

The nucleotide sequences of the present invention encoding ApoA-Ipeptides and agonists may be expressed on their own, in multimeric formor may be expressed as a chimeric with nucleotide sequences encodingnative or proform of ApoA-I.

The ApoA-I agonists of the invention are based, in part, on theApplicants' surprising discovery that altering certain amino acidresidues in the primary sequence of the 22-mer consensus sequence ofVenkatachalapathi et al., 1991, Mol. Conformation and Biol. InteractionsB:585-596 (PVLDEFREKLNEELEALKQKLK; SEQ ID NO: 75; hereinafter “Segrest'sconsensus 22-mer” or “consensus 22-mer”) that were thought to becritical for activity yields synthetic peptides that exhibit activitiesthat approach, or in some embodiments even exceed, the activity ofnative ApoA-I. In particular, the Applicants have discovered thatreplacing three charged amino acid residues in Segrest's consensus22-mer peptide (Glu-5, Lys-9 and Glu-13) with a hydrophobic. Leu residueprovides peptides that mimic the structural and functional properties ofApoA-I to a degree that is unprecedented in the art.

While not intending to be bound by any particular theory, it is believedthat the helix formed by the ApoA-I agonists of the invention moreclosely mimics the structural and functional properties of theamphipathic helical regions of native ApoA-I that are important foreffecting lipid-binding, cholesterol efflux and LCAT activation thandoes the α-helix formed by the ApoA-I mimetic peptides described in theliterature, thereby resulting in peptides that exhibit significantlyhigher ApoA-I-like activity than these other peptides. Indeed, whereasmany of the ApoA-I agonists of the invention approach, and in someembodiments even exceed, the activity of ApoA-I, to date, the bestpeptide ApoA-I mimics described in the literature—peptide 18AM4 (SEQ IDNO: 246); Corinjn et al., 1993, Biochim. Biophys. Acta 1170:8-16; Labeuret al., October 1994, Arteriosclerosis: Abstract Nos. 186 and 187; andN-acetylated, C-amidated peptide 18AM4 (SEQ ID NO: 239) (Brasseur, 1993,Biochim. Biophys. Acta 1170:1-7)—exhibit less than 4% and 11%,respectively, of the activity of ApoA-I as measured by the LCATactivation assay described herein.

In an illustrative embodiment of the invention, the nucleotide sequencesencode core peptides (or analogues thereof) that compose the ApoA-Iagonists of the invention having the following structural formula:(I)X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X5-X₁6-X₁₆X₁₇-X₁₈B-X₁₉-X₂₀-X₂₁-X₂₂  (I)wherein:

-   -   X₁ is Pro (P), Ala (A), Gly (G), Gln (Q), Asp (D) or Asn (N);    -   X₂ is an aliphatic amino acid;    -   X₃ is Leu (L) or Phe (F);    -   X₄ is an acidic amino acid;    -   X₅ is Leu (L) or Phe (F);    -   X₆ is Leu (L) or Phe (F);    -   X₇ is a hydrophilic amino acid;    -   X₈ is an acidic or a basic amino acid;    -   X₉ is Leu (L) or Gly (G);    -   X₁₀ is Leu (L), Trp (W) or Gly (G);    -   X₁₁ is a hydrophilic amino acid;    -   X₁₂ is a hydrophilic amino acid;    -   X₁₃ is Gly (G) or an aliphatic amino acid;    -   X₁₄ is Leu (L), Trp (W) or Gly (G);    -   X₁₅ is a hydrophilic amino acid;    -   X₁₆ is a hydrophobic amino acid;    -   X₁₇ is a hydrophobic amino acid;    -   X₁₈ is a basic amino acid, Gln (Q) or Asn (N);    -   X₁₉ is a basic amino acid, Gln (Q) or Asn (N);    -   X₂₀ is a basic amino acid;    -   X₂₁ is an aliphatic amino acid; and    -   X₂₂ is a basic amino acid.

In the core peptides of structure (I), the symbol “—” between amino acidresidues generally designates a ackbone constitutive linking function.Thus, the symbol “—” sually represents a peptide bond or amide linkage.

The present invention encompasses nucleotide sequences which encodepeptides which are functionally equivalent to the peptides of structure(I), for a more detailed description see U.S. patent application Ser.No. ______, page 25, line 6 to page 58, line 21, incorporated herein byreference in its entirety.

In a further illustrative embodiment of the present invention, thenucleotide sequences encode core peptides (or analogues thereof) thatcompose the ApoA-I agonists of the invention having the followingstructural formula:X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂  (II)wherein:

-   -   X₁ is Pro (P), Ala (A), Gly (G), Gln (Q), Asn (N) or Asp (D);    -   X₂ is an aliphatic amino acid;    -   X₃ is Leu (L) or Phe (F);    -   X₄ is Glu (E);    -   X₅ is an aliphatic amino acid;    -   X₆ is Leu (L) or Phe (F);    -   X₇ is Glu (E) or Leu (L);    -   X₈ is Asn (N) or Gln (Q);    -   X₉ is Leu (L);    -   X₁₀ is Leu (L), Trp (W) or Gly (G);    -   X₁₁ is an acidic amino acid;    -   X₁₂ is Arg (R);    -   X₁₃ is Leu (L) or Gly (G);    -   X₁₄ is Leu (L), Phe (F) or Gly (G);    -   X₁₅ is Asp (D);    -   X₁₆ is Ala (A);    -   X₁₇ is Leu (L);    -   X₁₈ is Asn (N) or Gln (Q);    -   X₁₉ is a basic amino acid;    -   X₂₀ is a basic amino acid;    -   X₂₁ is Leu (L); and    -   X₂₂ is a basic amino acid.

Each “—” independently designates a peptide bond or amide linkage.

The present invention encompasses nucleotide sequences which encodepeptides which are functionally equivalent to the peptides of structure(II), for a more detailed description see U.S. patent application Ser.No. ______, page 25, line 6 to page 58, line 21, incorporated herein byreference in its entirety.

In yet a further illustrative embodiment of the present invention, thenucleotide sequences encode core peptides (or analogues thereof) thatcompose the ApoA-I agonists of the invention having the followingstructural formula:X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈  (III)

-   -   X₁ is Pro (P), Ala (A), Gly (G), Asn (N) or Gln (Q);    -   X₂ is an aliphatic amino acid;    -   X₃ is Leu (L);    -   X₄ is an acidic amino acid;    -   X₅ is Leu (L) or Phe (F);    -   X₆ is Leu (L) or Phe (F);    -   X₇ is a basic amino acid;    -   X₈ is an acidic amino acid;    -   X₉ is Leu (L) or Trp (W);    -   X₁₀ is Leu (L) or Trp (W);    -   X₁₁ is an acidic amino acid or Asn (N);    -   X₁₂ is an acidic amino acid;    -   X₁₃ is Leu (L), Trp (W) or Phe (F);    -   X₁₄ is a basic amino acid or Leu (L);    -   X₁₅ is Gln (Q) or Asn (N);    -   X₁₆ is a basic amino acid;    -   X₁₇ is Leu (L); and    -   X₁₈ is a basic amino acid.

Each “—” independently designates a peptide bond or amide linkage.

The present invention encompasses nucleotide sequences which encodepeptides which are functionally equivalent to the peptides of structure(III), for a more detailed description, see U.S. patent application Ser.No.: ______, page 24, line 31 to page 58, line 3, incorporated herein byreference in its entirety.

The core peptides of structure (I), (II) and (III) are defined, in part,in terms of amino acids of designated classes. The definitions of thevarious designated classes are provided infra in connection with thedescription of mutated or altered embodiments of structure (I), (II) and(III).

All other letters in structures (I), (II) and (III) refer to theone-letter amino acid abbreviations commonly employed in the art, aspreviously described. The nucleotide sequence of the peptides may bededuced as follows: TABLE I SECOND BASE U C A G U

C

A

G

While not intending to be bound by any particular theory, it is believedthat the nucleotide sequences of the present invention encode peptideswhich form an amphipathic α-helix formed in the presence of lipids moreclosely mimics the structural and amphipathic properties of the helicalregions of native ApoA-I that are important for effecting lipid-binding,cholesterol efflux and LCAT activation than does the α-helix formed bythe peptides described in the literature, thereby resulting in peptidesthat exhibit significantly higher ApoA-I-like activity than these otherpeptides. Indeed, whereas many of the core peptides of structure (I),(II) and (III) approach, and in some embodiments even exceed, theactivity of ApoA-I, to date the best peptide ApoA-I mimic described inthe literature has less than 40% of the activity of ApoA-I as measuredby the LCAT activation assay described herein.

While not intending to be bound by any particular theory, it is believedthat certain structural and/or physical properties of the amphipathichelix formed by the core peptides of structure (I), (II) and (III) areimportant for activity. These properties include the degree ofamphipathicity, overall hydrophobicity, mean hydrophobicity, hydrophobicand hydrophilic angles, hydrophobic moment, mean hydrophobic moment, netcharge and dipole moment.

A summary of the preferred physical and structural properties of thecore peptides of structure (I), (II) and (III) is provided in TABLES II,III and IV below: TABLE II PHYSICAL PROPERTIES OF PREFERRED ApoA-IAGONISTS OF STRUCTURE (I) PROPERTY RANGE PREFERRED RANGE % hydrophobicamino 40-70 50-60 acids <H_(o)> −0.050 to −0.070 −0.030 to −0.055 <H_(o)^(pho)> 0.90-1.2  0.94-1.1  <μ_(H)> 0.45-0.65 0.50-0.60 pho angle160°-220° 180°-200° # positively 3-5 4 charged amino acids # negatively3-5 4 charged amino acids net charge −1 to +1 0 hydrophobic clusterpositions 3, 6, 9, 10 are hydrophobic amino acids acidic cluster atleast 1 acidic amino acid per turn except for last 5 C-terminal aminoacids basic cluster at least 3 basic amino acids in last 5 C- terminalamino acids

TABLE III PHYSICAL PROPERTIES OF PREFERRED ApoA-I AGONISTS OF STRUCTURE(II) PROPERTY RANGE PREFERRED RANGE % hydrophobic amino 40-70 50-60acids <H_(o)> −0.050 to −0.070 −0.030 to −0.055 <H_(o) ^(pho)> 0.90-1.2 0.94-1.1  <μ_(H)> 0.45-0.65 0.50-0.60 pho angle 160°-220° 180°-200° #positively 3-5 4 charged amino acids # negatively 3-5 4 charged aminoacids net charge −1 to +1 0 hydrophobic cluster positions 3, 6, 9, 10are hydrophobic amino acids acidic cluster at least 1 acidic amino acidper turn except for last 5 C-terminal amino acids basic cluster at least3 basic amino acids in last 5 C- terminal amino acids

TABLE IV PHYSICAL PROPERTIES OF PREFERRED ApoA-I AGONISTS OF STRUCTURE(III) PROPERTY RANGE PREFERRED RANGE % hydrophobic amino 40-70 50-60acids <H_(o)> −0.150 to −0.070 −0.130 to −0.050 <H_(o) ^(pho)> 0.90-1.2 0.95-1.10 <μ_(H)> 0.55-0.65 0.58-0.62 pho angle 120°-160° 130°-150° #positively 3-5 4 charged amino acids # negatively 3-5 4 charged aminoacids net charge −1 to +1 0 hydrophobic cluster positions 3, 6, 9, 10are hydrophobic amino acids acidic cluster at least 1 acidic amino acidper turn except for last 5 C-terminal amino acids basic cluster at least2 basic amino acids in last 5 C- terminal amino acids

The properties of the amphipathic α-helices formed by the core peptidesof the invention differ significantly from the properties of class Aamphipathic α-helices, particularly the class A α-helix of Segrest's 18Aand consensus 22-mer peptides. These differences are illustrated withexemplary core peptide 210 (SEQ ID NO: 210).

A comparison of the physical and structural properties of peptide 210(SEQ ID NO:210) and Segrest's 18A peptide (SEQ ID NO:244) and consensus22-mer peptide (SEQ ID NO:75) is provided in TABLE V, below: TABLE VCOMPARISON OF PROPERTIES OF EXEMPLARY CORE PEPTIDE 210 (SEQ ID NO: 210)WITH SEGREST'S CONSENSUS 22-MER (SEQ ID NO: 75) AND 18A PEPTIDE (SEQ IDNO: 244) CONSENSUS PROPERTY 18A 22-MER PEPTIDE 210 # amino acids 18 2218 # hydrophilic 9 13 9 amino acids # hydrophobic 9 9 9 amino acids %hydrophobic 50 41 50 amino acids <H_(o)> −0.43 −0.293 −0.125 <H_(o)^(pho)> 0.778 0.960 1.081 <μ_(H)> 0.485 0.425 0.597 pho angle 100° 100°140° # positively 4 5 4 charged amino acids # negatively 4 6 4 chargedamino acids net charge 0 −1 0

These differences in properties lead to significant differences inactivity. Whereas Segrest's 18A peptide (SEQ ID NO:244) and consensus22-mer peptide (SEQ ID NO:75) exhibit only 5% and 10% LCAT activation,respectively, as compared with native ApoA-I in the assays describedherein, peptide 210 (SEQ ID NO:210) exhibits 46% activation as comparedwith native ApoA-I in the same assays.

Certain amino acid residues in the core peptides of structure (I), (II)and (III) can be replaced with other amino acid residues withoutsignificantly deleteriously affecting, and in many cases even enhancing,the activity of the peptides. Thus, also contemplated by the presentinvention are mutated forms of the core peptides of structure (I), (II)and (III) wherein at least one defined amino acid residue in thestructure is substituted with another amino acid. As one of the criticalfeatures affecting the activity of the core peptides of the invention isbelieved to be their ability to form α-helices in the presence of lipidsthat exhibit the amphipathic and other properties described above, itwill be recognized that in preferred embodiments of the invention, theamino acid substitutions are conservative, i.e., the replacing aminoacid residue has physical and chemical properties that are similar tothe amino acid residue being replaced.

For purposes of determining conservative amino acid substitutions, theamino acids can be conveniently classified into two maincategories—hydrophilic and hydrophobic—depending primarily on thephysical-chemical characteristics of the amino acid side chain. Thesetwo main categories can be further classified into subcategories thatmore distinctly define the characteristics of the amino acid sidechains. For example, the class of hydrophilic amino acids can be furthersubdivided into acidic, basic and polar amino acids. The class ofhydrophobic amino acids can be further subdivided into apolar andaromatic amino acids. The definitions of the various categories of aminoacids that define structure (I), (II) and (III) are as follows:

“Hydrophophilic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of less than zero according to the consensushydrophobicity scale of Eisenberg, 1984, Ann. Rev. Biochem. 53:595-623.Genetically encoded hydrophilic amino acids include Pro (P), Thr (T),Ser (S), His (H), Glu (E), Asn (N), Gln (Q), Asp (D), Lys (K) and Arg(R).

“Acidic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of less than 7. Acidic amino acids typically havenegatively charged side chains at physiological pH due to loss of ahydrogen ion. Genetically encoded acidic amino acids include Glu (E) andAsp (D).

“Basic Amino Acid” refers to a hydrophilic amino acid having a sidechain pK value of greater than 7. Basic amino acids typically havepositively charged side chains at physiological pH due to associationwith hydronium ion. Genetically encoded basic amino acids include His(H), Arg (R) and Lys (K).

“Polar Amino Acid” refers to a hydrophilic amino acid having a sidechain that is uncharged at physiological pH, but which has at least onebond in which the pair of electrons shared in common by two atoms isheld more closely by one of the atoms. Genetically encoded polar aminoacids include Cys (C), Asn (N), Gln (Q) Ser (S) and Thr (T).

“Hydrophobic Amino Acid” refers to an amino acid exhibiting ahydrophobicity of greater than zero according to the consensushydrophobicity scale of Eisenberg, 1984, Ann. Rev. Biochem. 53:595-623.Genetically encoded hydrophobic amino acids include Ile (I), Phe (F),Val (V), Leu (L), Trp (W), Met (M), Ala (A), Gly (G) and Tyr (Y).

“Aromatic Amino Acid” refers to a hydrophobic amino acid with a sidechain having at least one aromatic or heteroaromatic ring. The aromaticor heteroaromatic ring may contain one or more substituents such as —OH,—SH, —CN, —F, —Cl, —Br, —I, —NO₂, —NO, —NH₂, —NHR, —NRR, —C(O)R,—C(O)OH, —C(O)OR, —C(O)NH₂, —C(O)NHR, —C(O)NRR and the like where each Ris independently (C₁-C₆) alkyl, substituted (C₁-C₆) alkyl, (C₁-C₆)alkenyl, substituted (C₁-C₆) alkenyl, (C₁-C₆) alkynyl, substituted(C₁-C₆) alkynyl, (C₅-C₂₀) aryl, substituted (C₅-C₂₀) aryl, (C₆-C₂₆)alkaryl, substituted (C₆-C₂₆) alkaryl, 5-20 membered heteroaryl orsubstituted 5-20 membered heteroaryl. Genetically encoded aromatic aminoacids include Phe (F), Tyr (Y) and Trp (W).

“Nonpolar Amino Acid” refers to a hydrophobic amino acid having a sidechain that is uncharged at physiological pH and which has bonds in whichthe pair of electrons shared in common by two atoms is generally heldequally by each of the two atoms (i.e., the side chain is not polar).Genetically encoded apolar amino acids include Leu (L), Val (V), Ile(I), Met (M), Gly (G) and Ala (A).

“Aliphatic Amino Acid” refers to a hydrophobic amino acid having analiphatic hydrocarbon side chain. Genetically encoded aliphatic aminoacids include Ala (A), Val (V), Leu (L) and Ile (I).

The amino acid residue Cys (C) is unusual in that it can form disulfidebridges with other Cys (C) residues or other sulfanyl-containing aminoacids. The ability of Cys (C) residues (and other amino acids with —SHcontaining side chains) to exist in a peptide in either the reduced free—SH or oxidized disulfide-bridged form affects whether Cys (C) residuescontribute net hydrophobic or hydrophilic character to a peptide. WhileCys (C) exhibits a hydrophobicity of 0.04 according to the consensusscale of Eisenberg (Eisenberg, 1984, supra), it is to be understood thatfor purposes of the present invention Cys (C) is categorized as a polarhydrophilic amino acid, notwithstanding the general classificationsdefined above.

As will be appreciated by those of skill in the art, the above-definedcategories are not mutually exclusive. Thus, amino acids having sidechains exhibiting two or more physical-chemical properties can beincluded in multiple categories. For example, amino acid side chainshaving aromatic moieties that are further substituted with polarsubstituents, such as Tyr (Y), may exhibit both aromatic hydrophobicproperties and polar or hydrophilic properties, and can therefore beincluded in both the aromatic and polar categories. The appropriatecategorization of any amino acid will be apparent to those of skill inthe art, especially in light of the detailed disclosure provided herein.

Certain amino acid residues, called “helix breaking” amino acids, have apropensity to disrupt the structure of α-helices when contained atinternal positions of the helix. Amino acid residues exhibitinghelix-breaking properties are well-known in the art (see, e.g., Chou andFasman, Ann. Rev. Biochem. 47:251-276) and include Pro (P), D-Pro (p)and Gly (G). While these amino acids conveniently fall into thecategories defined above, with the exception of Gly (G) these residuesshould not be used to substitute amino acids at internal positionswithin the helix—they should only be used to substitute 1-3 amino acidresidues at the N-terminus and/or C-terminus of the peptide.

The native structure of ApoA-I contains eight helical units that arethought to act in concert to bind lipids (Nakagawa et al., 1985, J. Am.Chem. Soc. 107:7087-7092; Anantharamaiah et al., 1985, J. Biol. Chem.260:10248-10262; Vanloo et al., 1991, J. Lipid Res. 32:1253-1264; Mendezet al., 1994, J. Clin. Invest. 94:1698-1705; Palgunari et al., 1996,Arterioscler. Thromb. Vasc. Biol. 16:328-338; Demoor et al., 1996, Eur.J. Biochem. 239:74-84). Thus, also included in the present invention areApoA-I agonists comprised of dimers, trimers, tetramers and even higherorder polymers (“multimers”) of the core peptides described herein. Suchmultimers may be in the form of tandem repeats, branched networks orcombinations thereof. The core peptides may be directly attached to oneanother or separated by one or more linkers.

The core peptides that comprise the multimers may be the peptides ofstructure (I), (II) or (III) analogues of structure (I), (II) or (III),mutated forms of structure (I), (II) or (III) truncated or internallydeleted forms of structure (I), (II) or (III) extended forms ofstructure (I), (II) or (III) and/or combinations thereof. The corepeptides can be connected in a head-to-tail fashion (i.e., N-terminus toC-terminus), a head-to-head fashion, (i.e., N-terminus to N-terminus), atail-to-tail fashion (i.e., C-terminus to C-terminus), or combinationsthereof.

In one embodiment of the invention, the multimers are tandem repeats oftwo, three, four and up to about ten core peptides. Preferably, themultimers are tandem repeats of from 2 to 8 core peptides. Thus, in oneembodiment, the ApoA-I agonists of the invention comprise multimershaving the following structural formula:HH-[LL_(m)-HH-]_(n)LL_(m)-HHwherein:

-   -   each m is independently an integer from 0 to 1, preferably 1;    -   n is an integer from 0 to 10, preferably 0 to 8;    -   each “HH” independently represents a core peptide or peptide        analogue of structure (I) or a mutated, truncated, internally        deleted or extended form thereof as described herein;    -   each “LL” independently represents a linker, e.g., Pro (P),        Gly (G) and Cys-Cys.

In a preferred embodiment of the invention, the tandem repeats areinternally punctuated by a single proline residue. To this end, in thoseinstances where the core peptides are terminated at their N- orC-terminus with proline, such as, e.g., where X₁ in structure (I) is Pro(P). In those instances where the core peptides do not contain an N- orC-terminal proline, LL is preferably Pro (P).

In certain embodiments of the invention, it may be desirable to employcleavable linkers that permit the release of one or more helicalsegments (HH) under certain conditions. Suitable cleavable linkersinclude peptides having amino acid sequences that are recognized byproteases, oligonucleotides that are cleaved by endonucleases.Preferably, the cleavage conditions will be relatively mild so as not todenature or otherwise degrade the helical segments and/or non-cleavedlinkers composing the multimeric ApoA-I agonists.

Peptide and oligonucleotide linkers that can be selectively cleaved, aswell as means for cleaving the linkers are well known and will bereadily apparent to those of skill in the art.

In a preferred embodiment, the linkers employed are peptides that aresubstrates for endogenous circulatory enzymes, thereby permitting themultimeric ApoA-I agonists to be selectively cleaved in vivo. Endogenousenzymes suitable for cleaving the linkers include, for example,proapolipoprotein A-I propeptidase. Appropriate enzymes, as well aspeptide segments that act as substrates for such enzymes, are well-knownin the art (see, e.g., Edelstein et al., 1983, J. Biol. Chem.258:11430-11433; Zanis, 1983, Proc. Natl. Acad. Sci. USA 80:2574-2578).

As discussed above, a key feature of the core peptides of the inventionis their ability to form intermolecular hydrogen-bonds or salt bridgeswhen arranged in an antiparallel fashion. Thus, in a preferredembodiment of the invention, linkers of sufficient length andflexibility are used so as to permit the helical segments (HH) ofstructure (II) to align in an antiparallel fashion and formintermolecular hydrogen-bonds or salt bridges in the presence of lipids.Linkers of sufficient length and flexibility include, but are notlimited to, Pro (P), Gly (G), Cys-Cys.

Alternatively, as the native apolipoproteins permit cooperative bindingbetween antiparallel helical segments, peptide linkers which correspondin primary sequence to the peptide segments connecting adjacent helicesof the native apolipoproteins, including, for example, ApoA-I, ApoA-II,ApoA-IV, ApoC-I, ApoC-II, ApoC-III, ApoD, ApoE and ApoJ can beconveniently used to link the core peptides. These sequences are wellknown in the art (see, e.g., Rosseneu et al., “Analysis of the Primaryand of the Secondary Structure of the Apolipoproteins,” In: Structureand Function of Lipoproteins, Ch. 6, 159-183, CRC Press, Inc., 1992).

Other linkers which permit the formation of intermolecular hydrogenbonds or salt bridges between tandem repeats of antiparallel helicalsegments include peptide reverse turns such as β-turns and γ-turns.Generally, reverse turns are segments of peptide that reverse thedirection of the polypeptide chain so as to allow a single polypeptidechain to adopt regions of antiparallel β-sheet or antiparallel α-helicalstructure. β-turns generally are composed of four amino acid residuesand γ-turns are generally composed of three amino acid residues.

The conformations and sequences of many peptide β-turns have beenwell-described in the art and include, by way of example and notlimitation, type-I, type-I′, type-II, type-II′, type-III, type-III′,type-IV, type-V, type-V′, type-VIa, type-VIb, type-VII and type-VIII(see, Richardson, 1981, Adv. Protein Chem. 34:167-339; Rose et al.,1985, Adv. Protein Chem. 37:1-109; Wilmot et al., 1988, J. Mol. Biol.203:221-232; Sibanda et al., 1989, J. Mol. Biol. 206:759-777; Tramontanoet al., 1989, Proteins: Struct. Funct. Genet. 6:382-394).

The specific conformations of short peptide turns such as β-turns dependprimarily on the positions of certain amino acid residues in the turn(usually Gly, Asn or Pro). Generally, the type-I β-turn is compatiblewith any amino acid residue at positions 1 through 4 of the turn, exceptthat Pro cannot occur at position 3. Gly predominates at position 4 andPro predominates at position 2 of both type-I and type-II turns. Asp,Asn, Ser and Cys residues frequently occur at position 1, where theirside chains often hydrogen-bond to the NH of residue 3.

In type-II turns, Gly and Asn occur most frequently at position 3, asthey adopt the required backbone angles most easily. Ideally, type-I′turns have Gly at positions 2 and 3, and type-II′ turns have Gly atposition 2. Type-III turns generally can have most amino acid residues,but type-III′ turns usually require Gly at positions 2 and 3. Type-VIaand VIb turns generally have a cis peptide bond and Pro as an internalresidue. For a review of the different types and sequences of β-turns inproteins and peptides the reader is referred to Wilmot et al., 1988, J.Mol. Biol. 203:221-232.

The conformation and sequences of many peptide γ-turns have also beenwell-described in the art (see, e.g., Rose et al., 1985, Adv. ProteinChem. 37:1-109; Wilmer-White et al., 1987, Trends Biochem. Sci.12:189-192; Wilmot et al., 1988, J. Mol. Biol. 203:221-232; Sibanda etal., 1989, J. Mol. Biol. 206:759-777; Tramontano et al., 1989, Proteins:Struct. Funct. Genet. 6:382-394). All of these types of β-turns andγ-turn structures and their corresponding sequences, as well as laterdiscovered peptide β-turns and γ-turn structures and sequences, arespecifically contemplated by the invention.

While structure (I), (II) and (III) contains 22 specified amino acidresidue positions, it is to be understood that the core peptides of theinvention can contain fewer than 22 amino acid residues. Indeed,truncated or internally deleted forms of structure (I), (II) and (III)containing as few as 18 or even 15 amino acid residues thatsubstantially retain the overall characteristics and properties of theamphipathic helix formed by the core peptides of structure (I), (II) and(III) are considered to be within the scope of the present invention.

Truncated forms of the peptides of structure (I) are obtained bydeleting one or more amino acids from the N- and/or C-terminus ofstructure (I), (II) and (III). Internally deleted forms of structure(I), (II) and (III) are obtained by deleting one or more amino acidsfrom internal positions within the peptide of structure (I), (II) and(III). The internal amino acid residues deleted may or may not beconsecutive residues.

Those of skill in the art will recognize that deleting an internal aminoacid residue from a core peptide of structure (I), (II) and (III) willcause the plane of the hydrophilic-hydrophobic interface of the helix torotate by 100° at the point of the deletion. As such rotations cansignificantly alter the amphipathic properties of the resultant helix,in a preferred embodiment of the invention amino acid residues aredeleted so as to substantially retain the alignment of the plane of thehydrophilic-hydrophobic interface along the entire long axis of thehelix.

This can be conveniently achieved by deleting a sufficient number ofconsecutive or non-consecutive amino acid residues such that onecomplete helical turn is deleted. An idealized α-helix contains 3.6residues per turn. Thus, in a preferred embodiment, groups of 3-4consecutive or non-consecutive amino acid residues are deleted. Whether3 amino acids or 4 amino acids are deleted will depend upon the positionwithin the helix of the first residue to be deleted. Determining theappropriate number of consecutive or non-consecutive amino acid residuesthat constitute one complete helical turn from any particular startingpoint within an amphipathic helix is well within the capabilities ofthose of skill in the art.

Due to the surmised importance of the basic cluster at the C-terminus ofthe core peptides of structure (I), (II) and (III) in stabilizing thehelix and the importance of the hydrophobic cluster in effecting lipidbinding and LCAT activation, in preferred embodiments of the invention,residues comprising the basic and hydrophobic clusters are not deleted.Thus, in preferred embodiments, residues 19, 20 and 22 (basic cluster)and residues 3, 6, 9 and 10 (hydrophobic cluster) are not deleted.

The core peptides of structure (I), (II) and (III) can also be extendedat one or both termini or internally with additional amino acid residuesthat do not substantially interfere with, and in some embodiments evenenhance, the structural and/or functional properties of the peptides.Indeed, extended core peptides containing as many as 23, 25, 26, 29 oreven more amino acid residues are considered to be within the scope ofthe present invention. Preferably, such extended peptides willsubstantially retain the net amphipathicity and other properties of thepeptides of structure (I), (II) and (III). Of course, it will berecognized that adding amino acids internally will rotate the plane ofthe hydrophobic-hydrophilic interface at the point of the insertion in amanner similar to that described above for internal deletions. Thus, theconsiderations discussed above in connection with internal deletionsapply to internal additions, as well.

In one embodiment, the core peptides are extended at the N- and/orC-terminus by least one helical turn. Preferably, such extensions willstabilize the helical secondary structure in the presence of lipids,such as the end-cap amino acids and segments previously described.

In a particularly preferred embodiment, the core peptide of structure(I), (II) and (III) is extended at the C-terminus by a single basicamino acid residue, preferably Lys (K).

5.1.3. PREFERRED EMBODIMENTS

The ApoA-I agonists encoded by the nucleotide sequences of the presentinvention can be further defined by way of preferred embodiments.

In one preferred embodiment, the nucleotide sequences of the inventionencode ApoA-I agonists composed of core peptides of structure (I)containing 22 amino acid residues in which:

-   -   X₁ is Pro (P), Gly (G) or Ala (A);    -   X₂ is Val (V) or Leu (L);    -   X₃ is Leu (L);    -   X₄ is Asp (D) or Glu (E);    -   X₅ is Leu (L) or Phe (F);    -   X₆ is Leu (L) or Phe (F);    -   X₇ is Lys (K) or Arg (R);    -   X₈ is Glu (E);    -   X₉ is Leu (L);    -   X₁₀ is Leu (L) or Trp (W);    -   X₁ is Asn (N) or Gln (Q);    -   X₁₂ is Glu (E);    -   X₁₃ is Leu (L);    -   X₁₄ is Leu (L) or Trp (W);    -   X₁₅ is Glu (E);    -   X₁₆ is Ala (A) or Trp (W);    -   X₁₇ is Leu (L);    -   one of X₁₈ or X₁₉ is Gln (Q) and the other is Lys (K);    -   X₂₀ is Lys (K);    -   X₂₁ is Leu (L); and    -   X₂₂ is Lys (K).

In yet another preferred embodiment, the nucleotide sequences of thepresent invention encode ApoA-I agonists composed of core peptides ofstructure (I) containing 22 amino acid residues in which:

-   -   X₁ is Pro (P), Gly (G) or Ala (A);    -   X₂ is Val (V);    -   X₃ is Leu (L);    -   X₄ is Asp (D) or Glu (E);    -   X₅ is Leu (L) or Phe (F);    -   X₆ is Phe (F);    -   X₇ is Arg (R);    -   X₈ is Glu (E);    -   X₉ is Leu (L);    -   X₁₀ is Leu (L) or Trp (W);    -   X₁₁ is Asn (N);    -   X₁₂ is Glu (E);    -   X₁₃ is Gly (G);    -   X₁₄ is Leu (L);    -   X₁₅ is Glu (E);    -   X₁₆ is Ala (A) or Trp (W);    -   X₁₇ is Leu (L);    -   X₁₈ is Lys (K);    -   X₁₉ is Gln (Q);    -   X₂₀ is Lys (K);    -   X₂₁ is Leu (L); and    -   X₂₂ is Lys (K).

Particularly preferred nucleotide sequences according to this aspect ofthe invention are those that encode core peptides selected from thegroup consisting of: peptide 3 PVLDLFRELLNEGLEALKQKLK; (SEQ ID NO: 3)peptide 13 GVLDLFRELLNEGLEALKQKLK; (SEQ ID NO: 13) peptide 138PVLDLFRELLNEGLEWLKQKLK; (SEQ ID NO: 138) peptide 139PVLDLFRELWNEGLEALKQKLK; (SEQ ID NO: 139) peptide 141PVLDFFRELLNEGLEALKQKLK; (SEQ ID NO: 141) peptide 142PVLELFRELLNEGLEALKQKLK. (SEQ ID NO: 142)

In still another preferred embodiment, the nucleotide sequences encodeApoA-I agonists which are 22-amino acid residue peptides according tostructure (I), in which X₉ is Gly (G) and each of X₁₀, X₁₃, X₁₄, X₁₆ andX₁₇ is other than Gly (G). A particularly preferred ApoA-I agonistaccording to this aspect of the invention is peptide 20:PVLDLFREGLNELLEALKQKLK. (SEQ ID NO: 20)

In still another preferred embodiment, the nucleotide sequences encodeApoA-I agonists which are 22-amino acid residue peptides according tostructure (I), in which X₁₀ is Gly (G) and each of X₉, X₁₃, X₁₄, X₁₆ andX₁₇ is other than Gly (G). A particularly preferred ApoA-I agonistaccording to this aspect of the invention is peptide 9:PVLDLFRELGNELLEALKQKLK. (SEQ ID NO: 9)

In still another preferred embodiment, the nucleotide sequences encodeApoA-I agonists which are 22-amino acid residue peptides according tostructure (I), in which X₁₄ is Gly (G) and each of X₉, X₁₀, X₁₃, X₁₆ andX₁₇ is other than Gly (G). A particularly preferred ApoA-I agonistaccording to this aspect of the invention is peptide 126:PVLDLFRELLNELGEALKQKLK. (SEQ ID NO: 26)

In still another preferred embodiment, the nucleotide sequences areApoA-I agonists which are 22-amino acid residue peptides according tostructure (I), in which X₁₆ is Gly (G) and each of X₉, X₁₀, X₁₃, X₁₄ andX₁₇ is other than Gly (G). A particularly preferred ApoA-I agonistaccording to this aspect of the invention is peptide 22:PVLDLFRELLNELLEGLKQKLK. (SEQ ID NO: 22)

In still another preferred embodiment, the nucleotide sequences areApoA-I agonists which are 22-amino acid residue peptides according tostructure (I), in which X₁₇ is Gly (G) and each of X₉, X₁₀, X₁₃, X₁₄ andX₁₆ is other than Gly (G). A particularly preferred ApoA-I agonistaccording to this aspect of the invention is peptide 12:PVLDLFRELLNELLEAGKQKLK. (SEQ ID NO: 12)

In yet another preferred embodiment, the nucleotide sequences are ApoA-Iagonists which are 22-amino acid residue peptides according to structure(I), in which each of X₉, X₁₀, X₁₃, X₁₄, X₁₆ and X₁₇ is other than Gly(G).

Agonists are 22-amino acid residue peptides according to structure (I),in which:

-   -   X₁ is Pro (P), Gly (G), Ala (A);    -   X₂ is Val (V) or Leu (L);    -   X₃ is Leu (L);    -   X₄ is Asp (D) or Glu (E);    -   X₅ is Leu (L) or Phe (F);    -   X₆ is Leu (L) or Phe (F);    -   X₇ is Arg (R) or Lys (K);    -   X₈ is Glu (E);    -   X₉ is Leu (L);    -   X₁₀ is Leu (L) or Trp (W);    -   X₁₁ is Asn (N) or Gln (Q);    -   X₁₂ is Glu (E);    -   X₁₃ is Leu (L);    -   X₁₄ is Leu (L) or Trp (W);    -   X₁₅ is Glu (E);    -   X₁₆ is Ala (A), Leu (L) or Trp (W);    -   X₁₇ is Leu (L);    -   one of X₁₈ or X₁₉ is Gln (Q) and the other is Lys (K);    -   X₂₀ is Lys (K);    -   X₂₁ is Leu (L); and    -   X₂₂ is Lys (K).

In a particularly preferred embodiment according to this aspect of theinvention, X₂ is Val (V); X₄ is Asp (D); X₅ is Leu (L); X₆ is Phe (F);X₇ is Arg R); X₁₀ is Leu (L); X₁₁ is Asn (N); X₁₃ is Leu (L); X₁₄ is Leu(L); X₁₆ is Ala (A) ; X₁₇ is Leu (L); X₁₈ is Lys (K); X₁₉ is Gln (Q);X₂₀ is Lys (K) and/or X₂₂ is Lys (K).

In still another preferred embodiment, the nucleotide sequences encodeApoA-I agonists which are altered or mutated forms of the peptides ofstructure (I) in which:

-   -   X₁ is other than Val (V) or Leu (L);    -   X₅ is other than Lys (K), Glu (E) or Trp (W);    -   X₆ is other than Trp (W);    -   X₇ is other than Trp (W) or Leu (L);    -   X₈ is other than Trp (W);    -   X₉ is other than Lys (K) or Trp (W);    -   X₁₁ is other than Trp (W);    -   X₁₂ is other than Trp (W) or Leu (L);    -   X₁₃ is other than Glu (E) or Trp (W);    -   X₁₅ is other than Trp (W); and/or    -   X₂₁ is other than Lys (K).

In yet another preferred embodiment, the nucleotide sequences of theinvention encode ApoA-I agonists which are selected from the group ofpeptides set forth below: peptide 2 GVLDLFRELLNELLEALKQKLKK; (SEQ ID NO:2) peptide 3 PVLDLFRELLNELLEWLKQKLK; (SEQ ID NO: 3) peptide 4PVLDLFRELLNELLEALKQKLK; (SEQ ID NO: 4) peptide 7 PVLDLFKELLNELLEALKQKLK;(SEQ ID NO: 7) peptide 8 PVLDLFRELLNEGLEALKQKLK; (SEQ ID NO: 7) peptide9 PVLDLFRELGNELLEALKQKLK; (SEQ ID NO: 8) peptide 11PVLDLFKELLQELLEALKQKLK; (SEQ ID NO: 9) peptide 12PVLDLFRELLNELLEAGKQKLK; (SEQ ID NO: 10) peptide 13GVLDLFRELLNEGLEALKQKLK; (SEQ ID NO: 11) peptide 15PVLDLFRELWNELLEALKQKLK; (SEQ ID NO: 12) peptide 16PVLDLLRELLNELLEALKQKLK; (SEQ ID NO: 13) peptide 17PVLELFKELLQELLEALKQKLK; (SEQ ID NO: 14) peptide 18GVLDLFRELLNELLEALKQKLK; (SEQ ID NO: 15) peptide 20PVLDLFREGLNELLEALKQKLK; (SEQ ID NO: 16) peptide 22PVLDLFRELLNELLEGLKQKLK; (SEQ ID NO: 17) peptide 23PLLELFKELLQELLEALKQKLK; (SEQ ID NO: 18) peptide 24PVLDLFRELLNELLEALQKKLK; (SEQ ID NO: 19) peptide 26PVLDLFRELLNELLELLKQKLK; (SEQ ID NO: 21) peptide 28PVLDLFRELLNELWEALKQKLK; (SEQ ID NO: 23) peptide 29AVLDLFRELLNELLEALKQKLK; (SEQ ID NO: 24) peptide 123QVLDLFRELLNELLEALKQKLK; (SEQ ID NO: 123) peptide 125NVLDLFRELLNELLEALKQKLK; (SEQ ID NO: 124) peptide 126PVLDLFRELLNELGEALKQKLK; (SEQ ID NO: 125) peptide 127PVLDLFRELLNELLELLKQKLK; (SEQ ID NO: 126) peptide 128PVLDLFRELLNELLEFLKQKLK; (SEQ ID NO: 127) peptide 129PVLELFNDLLRELLEALQKKLK; (SEQ ID NO: 128) peptide 130PVLELFNDLLRELLEALKQKLK; (SEQ ID NO: 129) peptide 131PVLELFKELLNELLDALRQKLK; (SEQ ID NO: 130) peptide 132PVLDLFRELLENLLEALQKKLK; (SEQ ID NO: 131) peptide 133PVLELFERLLEDLLQALNKKLK; (SEQ ID NO: 132) peptide 134PVLELFERLLEDLLKALNQKLK; (SEQ ID NO: 133) peptide 135DVLDLFRELLNELLEALKQKLK; (SEQ ID NO: 134) peptide 136PALELFKDLLQELLEALKQKLK; (SEQ ID NO: 135) peptide 138PVLDLFRELLNEGLEWLKQKLK; (SEQ ID NO: 136) peptide 139PVLDLFRELWNEGLEALKQKLK; (SEQ ID NO: 137) peptide 141PVLDFFRELLNEGLEALKQKLK; (SEQ ID NO: 138) peptide 142PVLELFRELLNEGLEALKQKLK. (SEQ ID NO: 139)

In preferred embodiment, the nucleotide sequences encode ApoA-I agonistswhich are 22 amino acid residue peptides according to structure (II), inwhich:

-   -   X₁ is Pro (P), Gly (G), Ala (A) or Asn (N);    -   X₂ is Ala (A), Val (V) or Leu (L);    -   X₅ is Leu (L);    -   X₆ is Phe (F);    -   X₁₁ is Glu (E);    -   X₁₉ is Lys (K);    -   X₂₀ is Lys (K); and/or    -   X₂₂ is Lys (K), and each of X₃, X₄, X₇, X₈, X₉, X₁₀, X₁₂, X₁₃,        X₁₄, X₁₅, X₁₆, X₁₇, X₁₈ and X₂₁ are as previously defined for        structure (II).

Particularly preferred ApoA-I agonists according to this aspect of theinvention are those in which X₂ is Val (V); and/or X₁₈ is Gln (Q).

In still another preferred embodiment, the ApoA-I agonists are 22 aminoacid residue peptides according to structure (II), in which one of X₁₀,X₁₃ or X₁₄ is Gly (G) and the others of X₁₀, X₁₃ and X₁₄ are other thanGly (G). When X₁₄ is Gly (G), X₇ is preferably Glu (E).

Particularly preferred nucleotide sequences according to this aspect ofthe invention encode ApoA-I agonists which are peptides selected fromthe group consisting of: peptide 148: PVLELFENLLERLGDALQKKLK; (SEQ IDNO: 148) peptide 151: PVLELFENLGERLLDALQKKLK; (SEQ ID NO: 151) peptide154: PVLELFENLLERGLDALQKKLK. (SEQ ID NO: 154)

In still another preferred embodiment, the ApoA-I agonists are 22-aminoacid residue peptides according to structure (II), in which each of X₁₀,X₁₃ and X₁₄ is other than Gly (G).

In still another preferred embodiment, the nucleotide sequences encodeApoA-I agonists which are altered or mutated forms of the peptidesaccording to structure (II), in which:

-   -   X₄ is other than Asp (D);    -   X₅ is other than Phe (F);    -   X₆ is other than Trp (W);    -   X₇ is other than Leu (L) or Asp (D);    -   X₉ is other than Gly (G) or Trp (W);    -   X₁₂ is other than Lys (K);    -   X₁₃ is other than Trp (W);    -   X₁₄ is other than Trp (W);    -   X₁₅ is other than Glu (E);    -   X₁₆ is other than Trp (W) or Leu (L); and/or    -   X₁₇ is other than Trp (W).

In still another preferred embodiment, the ApoA-I agonists are 22 aminoacid residue peptides according to structure (II), in which when X₇ isLeu (L), X₁₀ is Trp (W), X₁ is other than Gly (G) and/or X₁₄ is otherthan Gly (G). A particularly preferred peptide according to this aspectof the invention is peptide 155 (PVLELFLNLWERLLDALQKKLK; SEQ ID NO:155).

In still another preferred embodiment, the nucleotide sequences encodeApoA-I agonists which are selected from the group of peptides set forthbelow: peptide 145: GVLELFENLLERLLDALQKKLK; (SEQ ID NO: 145) peptide146: PVLELFENLLERLLDALQKKLK; (SEQ ID NO: 146) peptide 147:PVLELFENLLERLFDALQKKLK; (SEQ ID NO: 147) peptide 148:PVLELFENLLERLGDALQKKLK; (SEQ ID NO: 148) peptide 149:PVLELFENLWERLLDALQKKLK; (SEQ ID NO: 149) peptide 150:PLLELFENLLERLLDALQKKLK; (SEQ ID NO: 150) peptide 151:PVLELFENLGERLLDALQKKLK; (SEQ ID NO: 151) peptide 152:PVFELFENLLERLLDALQKKLK; (SEQ ID NO: 152) peptide 153:AVLELFENLLERLLDALQKKLK; (SEQ ID NO: 153) peptide 154:PVLELFENLLERGLDALQKKLK; (SEQ ID NO: 154) peptide 155:PVLELFLNLWERLLDALQKKLK; (SEQ ID NO: 155) peptide 186:PVLELFEQLLERLLDALQKKLK; (SEQ ID NO: 186) peptide 187:PVLELFENLLERLLDALNKKLK; (SEQ ID NO: 187) peptide 188:PVLELFENLLDRLLDALQKKLK; (SEQ ID NO: 188) peptide 189:DVLELFENLLERLLDALQKKLK. (SEQ ID NO: 189)

In one preferred embodiment, the ApoA-I agonists are 18 amino acidresidue peptides according to structure (III).

In another preferred embodiment, the nucleotide sequences encode ApoA-Iagonists which are 18 amino acid residue peptides according to structure(III) in which:

-   -   X₂ is Ala (A), Val (V) or Leu (L);    -   X₄ is Asp (D) or Glu (E);    -   X₇ is Arg (R) or Lys (K);    -   X₈ is Asp (D) or Glu (E);    -   X₁₁ is Glu (E) or Asn (N);    -   X₁₂ is Glu (E);    -   X₁₄ is Arg (R), Lys (K) or Leu (L);    -   X₁₆ is Arg (R) or Lys (K); and/or    -   X₁₈ is Arg (R) or Lys (K) and        X₁, X₃, X₅, X₆, X₉, X₁₀, X₁₃, X₁₅ and X₁₇ are as previously        defined for structure (III).

In another preferred embodiment, the ApoA-I agonists are 18 amino acidresidue peptides according to structure (III), in which when X₁₁ is Asn(N), X₁₄ is Leu (L) and when X₁₁ is other than Asn (N), X₁₄ is otherthan Leu (L). An exemplary particularly preferred embodiment accordingto this aspect of the invention is the peptide 209 (PVLDLFRELLNELLQKLK;SEQ ID NO: 209).

In still another preferred embodiment, the ApoA-I agonists are alteredor mutated forms of the peptides according to structure (III), in which

-   -   X₁ is other than Asp (D);    -   X₉ is other than Gly (G);    -   X₁₀ is other than Gly (G);    -   X₁₂ is other than Leu (L); and    -   X₁₃ is other than Gly (G).

In still another preferred embodiment, the nucleotide sequences encodeApoA-I agonists which are selected from the group of peptides set forthbelow: peptide 191 PVLDLLRELLEELKQKLK*; (SEQ ID NO: 191) peptide 192PVLDLFKELLEELKQKLK*; (SEQ ID NO: 192) peptide 193 PVLDLFRELLEELKQKLK*;(SEQ ID NO: 193) peptide 194 PVLELFRELLEELKQKLK*; (SEQ ID NO: 194)peptide 195 PVLELFKELLEELKQKLK*; (SEQ ID NO: 195) peptide 196PVLDLFRELLEELKNKLK*; (SEQ ID NO: 196) peptide 197 PLLDLFRELLEELKQKLK*;(SEQ ID NO: 197) peptide 198 GVLDLFRELLEELKQKLK*; (SEQ ID NO: 198)peptide 199 PVLDLFRELWEELKQKLK*; (SEQ ID NO: 199) peptide 200NVLDLFRELLEELKQKLK*; (SEQ ID NO: 200) peptide 201 PLLDLFKELLEELKQKLK*;(SEQ ID NO: 201) peptide 202 PALELFKDLLEELRQKLR*; (SEQ ID NO: 202)peptide 203 AVLDLFRELLEELKQKLK*; (SEQ ID NO: 203) peptide 204PVLDFFRELLEELKQKLK*; (SEQ ID NO: 204) peptide 205 PVLDLFREWLEELKQKLK*;(SEQ ID NO: 205) peptide 206 PLLELLKELLEELKQKLK*; (SEQ ID NO: 206)peptide 207 PVLELLKELLEELKQKLK*; (SEQ ID NO: 207) peptide 208PALELFKDLLEELRQRLK*; (SEQ ID NO: 208) peptide 209 PVLDLFRELLNELLQKLK;(SEQ ID NO: 209) peptide 210 PVLDLFRELLEELKQKLK; (SEQ ID NO: 210)peptide 213 PALELFKDLLEEFRQRLK*; (SEQ ID NO: 213) peptide 215PVLDLFRELLEEWKQKLK*; (SEQ ID NO: 215) peptide 229 PVLELFERLLEDLQKKLK;(SEQ ID NO: 229) peptide 230 PVLDLFRELLEKLEQKLK; (SEQ ID NO: 230)peptide 231 PLLELFKELLEELKQKLK*. (SEQ ID NO: 231)

In yet another preferred embodiment, the nucleotide sequences of thepresent invention encode ApoA-I agonists which are multimeric formsaccording to structure IV in which each HH is independently a peptideaccording to structure (I), (II) or (III), or any of the preferredpeptides according to structure (I), (II) or (III) described herein.

In a final preferred embodiment, the ApoA-I agonists are not any of thepeptides listed in Table VIII which are composed of amino acids notgenetically encoded or which exhibit an LCAT activation activity of lessthan 38% as compared with native human ApoA-I.

5.1.4. ANALYSIS OF STRUCTURE AND FUNCTION

The structure and function of the core peptides or peptide analogues ofthe invention, as well as ApoA-I agonists composed of such corepeptides, including the multimeric forms described above, can be assayedin order to select active agonists or mimetics of ApoA-I. For example,the core peptides or peptide analogues can be assayed for their abilityto form α-helices in the presence of lipids, to bind lipids, to formcomplexes with lipids, to activate LCAT, to promote cholesterol efflux,etc.

Methods and assays for analyzing the structure and/or function of thepeptides are well-known in the art. Preferred methods are provided inthe working examples, infra. For example, the circular dichroism (CD)and nuclear magnetic resonance (NMR) assays described in Section 7,infra, can be used to analyze the structure of the peptides or peptideanalogues—particularly the degree of helicity in the presence of lipids.The ability to bind lipids can be determined using the fluorescencespectroscopy assay described in Section 7, infra. The ability of thepeptides and/or peptide analogues to activate LCAT can be readilydetermined using the LCAT activation described in Section 8, infra. Thein vitro and in vivo assays described in Section 9 and 10, infra, can beused to evaluate the half-life, distribution, cholesterol efflux andeffects on RCT.

Generally, core peptides and/or peptide analogues according to theinvention which exhibit the properties listed in TABLE VI, infra, areconsidered to be active. TABLE VI PROPERTIES OF ACTIVE PEPTIDESPREFERRED PROPERTY RANGE RANGE % Helicity in the presence of ≧60% ≧80%lipids (Ri = 30) (unblocked 22- amino acid residue peptides) % Helicityin the presence of ≧40% ≧60% lipids (Ri = 30) (unblocked 18- amino acidresidue peptides) % Helicity in the presence of ≧60% ≧80% lipids (Ri =30) (blocked 18-amino acid residue peptides and shorter peptides) LipidBinding (in the presence of 0.5-10 μM SUVs) peptide R_(i) = 1-50 LCATactivation ≧38% ≧80%R_(i) is lipid:peptide molar ratio.

As illustrated in the working examples, infra, core peptides whichexhibit a high degree of LCAT activation (≧38%) generally possesssignificant α-helical structure in the presence of lipidic smallunilamellar vesicles (SUVs) (≧60% helical structure in the case ofunblocked peptides containing 22 or more amino acid residues and blockedpeptides containing 18 or fewer amino acid residues; ≧40% helicalstructure in the case of unblocked peptides containing 18 or fewer aminoacids), and those peptides which exhibit little or no LCAT activationpossess little α-helical structure. However, in certain instances,peptides which exhibit significant helical structure in the presence oflipids do not effect significant LCAT.

Similarly, while core peptides that exhibit significant LCAT activationtypically bind lipids, in certain instances peptides which exhibit lipidbinding do not effect significant LCAT activation.

As a consequence, it will be recognized by those of skill in the artthat while the ability of the core peptides described herein to formα-helices (in the presence of lipids) and to bind lipids is critical foractivity, in many instances these properties may not be sufficient.Thus, in a preferred embodiment core peptides of the invention aresubjected to a series of screens to select for core peptides exhibitingsignificant pharmacological activity.

In a first step, a core peptide is screened for its ability to form anα-helix in the presence of lipids using the CD assay described inSection 7, infra. Those peptides which are at least 40% helical or 60%helical in the presence of lipids (at a conc. of about 5 μM and alipid:peptide molar ratio of about 30) are then screened for theirability to bind lipids using the fluorescence assay described in Section7, infra. Of course, only those core peptides which contain afluorescent Trp (W) residue are screened for lipid binding viafluorescence. However, for peptides which do not contain fluorescentresidues, binding to lipids is obvious when helicity increases in thepresence of lipids.

Core peptides which exhibit lipid binding in the presence of SUVs(0.5-10 μM peptide; lipid:peptide molar ratio in the range of 1 to 50)are then screened for pharmacological activity. Of course, thepharmacological activity screened for will depend upon the desired useof the ApoA-I agonists. In a preferred embodiment, the core peptides arescreened for their ability to activate LCAT, as peptides which activateLCAT are particularly useful in the methods described herein. Corepeptides which exhibit at least about 38% LCAT activation as comparedwith native human ApoA-I (as determined using the LCAT activation assaydescribed in Section 8, infra), are preferred, with core peptidesexhibiting 50%, 60%, 70%, 80% or even 90% or more being particularlypreferred.

5.2. DNA VECTORS AND CASSETTES ENCODING ApoA-1

In accordance with the present invention the nucleotide sequencesencoding native ApoA-I, modified forms of ApoA-I or peptides with ApoA-Iactivity, including ApoA-I agonists and superagonists, are inserted intoa cassette or an appropriate expression vehicle, i.e., a vector whichcontains the necessary elements for the transcription and translation ofthe inserted coding sequence, or in the case of an RNA viral vector, thenecessary elements for replication and translation. The nucleotidesequences of the present invention may be administered as “naked” DNAconstructs for both ex vivo and in vivo gene therapy protocols. NakedDNA plasmids encoding the ApoA-I peptides and agonists may be injectedinto a subject and successfully taken up by cells and expressed aspeptides (Felgner et al. U.S. Pat. No. 5,580,859, incorporated herein byreference in its entirety). “Naked” DNA may also be administered incomplexes with nonlipid cationic polymers or in complexes with liposomesto enhance cellular uptake.

In another embodiment of the present invention the expression vehicle istransfected into a suitable target cell which will express the peptide.Depending on the expression system used, the expressed peptide is thenisolated by procedures well-established in the art. Methods forrecombinant protein and peptide production are well known in the art(see, e.g., Maniatis et al., 1989, Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory, N.Y.; and Ausubel et al., 1989,Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, N.Y.).

In accordance with the present invention, the DNA cassettes containnucleotide sequences encoding the preproform and the proform of ApoA-Ito ensure that the ApoA-I peptides are correctly processed and secretedfrom the host cell. The preproform of ApoA-I contains an 18aa leader orsignal sequence at the amino terminus cleaved upon secretion of theProApoA-I from the host cell. The leader sequence of ApoA-I is of thestandard length and hydrophobicity (see, Davis et al. 1980 Nature283:433-438) and ends in an amino acid with a small side chain, e.g.,alanine. By way of example and not by limitation, the following leadersequences may be incorporated into the DNA cassettes and vectors of thepresent invention: 5′MKAAVLTLAVLFLTGSQA3′ (SEQ ID NO: ______) or5′MKAAVLAVALVFLTGCQA3′ (SEQ ID NO: ______). Any sequence which resultsin the secretion of the peptide from the host cell may be used inaccordance with the present invention.

The proform of ApoA-I, ProApoA-I contains a six-amino acidamino-terminal extension with the.sequence: R-H-F-W-Q-Q (SEQ ID NO:______) or X-E-F-X-Q-Q. (SEQ ID NO: ______). The extracellular cleavageof the prosegment by a specific protease generates the plasma form ofApoA-I. In accordance with the present invention, the sequences encodingthe six-amino acid extension may be incorporated into the DNA cassettesand vectors of the invention to ensure that the encoded ApoA-I peptidesare correctly processed once they are secreted from the host cell.

To increase efficiency of production, the polynucleotide can be designedto encode multiple units of the peptide separated by enzymatic cleavagesites—either homopolymers (repeating peptide units) or heteropolymers(different peptides strung together) can be engineered in this way. Theresulting polypeptide can be cleaved (e.g., by treatment with theappropriate enzyme) in order to recover the peptide units. This canincrease the yield of peptides driven by a single promoter. In apreferred embodiment, a polycistronic polynucleotide can be designed sothat a single mRNA is transcribed which encodes multiple peptides (i.e.,homopolymers or heteropolymers) each coding region operatively linked toa cap-independent translation control sequence; e.g., an internalribosome entry site (IRES), such as the sequences described by Pelletieret al. 1988 Nature 334:320-325. In a preferred embodiment, the IRES isderived from the 5′ noncoding region of the human immunoglobulinheavy-chain-binding protein (BiP) mRNA (Macejak et al. 1991 Nature 353:90-94). Preferably, the IRES region is derived from a picornavirus IRESregion sequence; the IRES sequence is selected from the group consistingof an enterovirus, rhinovirus, cardiovirus, and aphthovirus IRESsequence; a hepatitis A virus IRES sequence, a hepatitis B virussequence and a hepatitis C virus IRES sequence. When used in appropriateviral expression systems, the translation of each peptide encoded by themRNA is directed internally in the transcript; e.g., by the IRES. Thus,the polycistronic construct directs the transcription of a single, largepolycistronic mRNA which, in turn, directs the translation of multiple,individual peptides. This approach eliminates the production andenzymatic processing of polyproteins and may significantly increaseyield of peptide driven by a single promoter.

A variety of host-expression vector systems may be utilized to expressthe peptides described herein. Any host-expression system may be used inaccordance with the present invention that the system (1) expresses theApoA-I peptides of the invention; (2) utilizes expression controlelements (e.g., promoters and enhancers) that are operable in mammaliancells; and (3) expresses the ApoA-I nucleotide sequences at high copynumbers. In a preferred embodiment, any expression system which resultin an increased copy number of the nucleotide sequences encoding ApoA-Ipeptides and agonists in human cells when engineered using ex vivo or invivo gene therapy protocols. These include, but are not limited to,microorganisms such as bacteria transformed with recombinantbacteriophage DNA or plasmid DNA expression vectors containing anappropriate coding sequence; yeast or filamentous fungi transformed withrecombinant yeast or fungi expression vectors containing an appropriatecoding sequence; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing an appropriate codingsequence; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing an appropriate coding sequence; or animal cellsystems.

The expression elements of the expression systems vary in their strengthand specificities. Depending on the host/vector system utilized, any ofa number of transcription and translation elements operable inmammalian, preferably human, host cell systems, including constitutiveand inducible promoters, may be used in the expression vector. In apreferred embodiment, the expression elements incorporated into thevectors and cassettes of the present invention are the cis- andtrans-regulatory elements which regulate expression of the native ApoA-Igene. In particular, transcriptional regulatory and enhancer elementsare found in the nucleotide sequences located −222 to −110 upstream ofthe ApoA-I gene. For example, the binding site for ApoA-I regulatoryprotein-1 (ARP-1) a member of the steroid hormone receptor superfamilyis within the liver-specific transcriptional enhancer, located −222 to−110 DNA region upstream of the ApoA-I gene. The consensus sequence forARP-1 binding is 5′TGAACCCTTGACCCCT3′ (SEQ ID NO: ______) (see, Ladiaset al. 1991 Science 251:561-565). Additional ApoA-I transcriptionalregulation sequences and enhancer elements are disclosed in Sorci-Thomaset al. 1991 Journal of Biol. Chem. 266:18045-18050, Dai et al. 1990 Eur.J. Biochem. 190:305-310, Rottman et al. 1991 Molecular and Cell Biology11:3814-3820.

When cloning in bacterial, plant, insect or mammalian cell systems,promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5 K promoter) may beused; when generating cell lines that contain multiple copies ofexpression product, SV40-, BPV- and EBV-based vectors may be used withan appropriate selectable marker.

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the ApoA-I peptide coding sequence of interest may be ligated toan adenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing an ApoA-I peptide in infected hosts. (e.g., See Logan andShenk, 1984, Proc. Natl. Acad. Sci. USA 81, 3655-3659). Specificinitiation signals may also be required for efficient translation ofinserted ApoA-I peptide coding sequences. These signals include the ATGinitiation codon and adjacent sequences. In cases where an entireApoA-I, including its own initiation codon and adjacent sequences, isinserted into the appropriate expression vector, no additionaltranslational control signals may be needed. However, in cases whereonly a portion of the ApoA-I peptide coding sequence is inserted,exogenous translational control signals, including, perhaps, the ATGinitiation codon, must be provided. Furthermore, the initiation codonmust be in phase with the reading frame of the desired coding sequenceto ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner, et al., 1987,Methods in Enzymol. 153, 516-544).

Other expression systems for producing the peptides of the inventionwill be apparent to those having skill in the art.

5.2.1. VIRAL VECTORS ENCODING ApoA-I

In accordance with the present invention, the nucleotide sequences andDNA cassettes described above encoding ApoA-I agonists or peptides withApoA-I activity may be engineered into appropriate viral vectors. Theviral vectors described herein are particularly useful for in vivogenetic approaches to supply the nucleotide sequences of the presentinvention. In accordance with the present invention, the viral vectorsengineered to express native ApoA-I are not encompassed by the presentinvention.

For the practice of the present invention, viruses which display asuitable tropism for disease target cells, e.g., liver cells, cells oflarge and small intestine, endothelial cells, etc., or which aregenetically engineered to display a suitable tropism for the targetcells, are genetically engineered to contain nucleotide sequences thatencode ApoA-I agonist peptides. For the practice of the presentinvention, viruses which display a tropism for liver cells and cells ofthe small and large intestine are a particularly preferred embodiment ofthe present invention. Viral vectors which can be used in accordancewith the invention include, but are not limited to hepadnavirus,adenovirus, adeno-associated virus, herpes virus, retrovirus,parvovirus, vaccinia virus, etc.

In a specific embodiment, attenuated viruses, such as hepadnaviruses,which have a natural tropism for hepatocytes, may be engineered and usedfor gene therapy in accordance with the present invention.Hepadnaviruses may be engineered to deliver the nucleotide sequencesencoding native ApoA-I and ApoA-I to the cells and organs where it isnaturally expressed. There are regions of the hepadnavirus genome whichcan serve as regions to insert foreign DNA sequences. In particular,sequences encoding the hepatitis B virus (HBV) surface antigen proteins,pre-S1 and pre-S2, can be targeted for the insertion of DNA sequencesencoding ApoA-I peptides and agonists. For example, nucleotide sequencesencoding the ApoA-I peptides may be insert downstream of the TATA-likesequence (at position 278) which is the promoter for the pre-S1 mRNA ormay be inserted downstream of the SV40 like promoter (at position 3166)which is the promoter for the pre-S2 mRNA. The insertion of foreigncoding sequences downstream of these regions places them under theregulation of viral gene expression without disruption of otheressential viral activities.

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes toliver and respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, 1993,Current opinion in Genetics and Development 3:499-503 present a reviewof adenovirus-based gene therapy. In addition, Gerard et al., 1996,Current Opin. in Lipidology 7:105-111 describes adenovirus-mediated genetransfer as a means of delivering ApoA-I. Bout et al., 1994, Human GeneTherapy 5:3-10 demonstrated the use of adenovirus vectors to transfergenes to the respiratory epithelia of rhesus monkeys. Other instances ofthe use of adenoviruses in gene therapy can be found in Rosenfeld etal., 1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;and Mastrangeli et al., 1993, J. Clin. Invest, 91:225-234.Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med. 204:289-300.

For example, the cassettes encoding heteropolymers of ApoA-I peptides(i.e., heteropolymers containing ApoA-I agonists and superagonistsseparated by enzyme cleavage sites or by IRES sequences) may beadvantageously engineered into adenoviruses. Gene sequences required forregulating adenoviral gene expression which can be manipulated inaccordance with the invention include region E1, region E3, or betweenthe right ITR and region E4. These regions may be replaced with the TREof interest and incorporated into viral particles by packaging of therecombinant adenoviral genome.

There are three regions of adenovirus which would be preferred sites forthe insertion of foreign DNA sequences for the purpose of engineeringrecombinant viruses. These regions include (a) the EIA region and themajor late promoter (MLP); (2) the MLP, together with the tripartiteleader; and (3) the replacement of the E3 region. The E3 region has beenshown to be dispensable for viral packaging in tissue culture. Chimericviruses resulting from insertion of foreign DNA sequences in the EIA andMLP regions may be rescued by growth in 293 cells or be co-transfectionwith a helper virus providing the missing function in trans.

The herpes simplex virus which displays a natural tropism for thenervous system, may be modified to infect hepatocytes or endothelialcells and to contain desired nucleotide sequences that encode ApoA-Ipeptides. For example, the cassettes encoding heteropolymers of ApoA-Ipeptides may be advantageously engineered downstream of an earlypromoter region of HSV such as the ICP4 gene sequences or the VP16 genesequences in order to enhance expression of these sequences. Severalstructural genes of HSV can be targeted for the insertion of foreign DNAsequences for the purpose of engineering recombinant viruses. Forexample, the nucleotide sequences encoding the structural glycoproteins,gB, gD and gH can serve as regions to insert foreign DNA sequences.However, those gene products which have been replaced and/or disruptedwould be supplied in trans, either by vector and/or expressed by hostcells in order to produce virus stocks of the resulting recombinantviruses.

Human retroviruses may be used to, e.g. HTLV-1 and HTLV-2 or animalretroviruses which display a tropism for human lymphocytes, e.g., Bovineleukemia virus, Moloney murine leukemia virus, Rous-associated virus,and feline leukemia virus, may be used to target liver cells. Sequencesrequired for retroviral replication are located in the LTR, therefore,foreign DNA sequences, i.e., coding sequences of the ApoA-I peptide ofinterest may be inserted downstream. In retrovirus-infected cells, viraltranscription occurs from the integrated viral DNAs, the provirus, whichrepresents a transcriptional unit that contains its own regulatorysequences. Expression of the provirus depends on (1) the site into whichthe provirus has integrated; (2) the physiological state of the cell;and (3) the viral LTR. The expression of the provirus depends almostentirely on host-encoded enzymes.

The viral LTR is derived from three segments of the viral RNA: the R, U5and U3 sequences. The R or redundant sequence is a short segment (30 to60 nucleotides) that is present twice in the viral RNA, at the extremeleft (5′) and right (3′) termini. The U5 sequences are present at the 5′end of the viral RNA. The U3 sequences are located upstream from the Rsequences and have the most variation in size (0.2 to 1.2 kb) andsequence homology.

Viral specific regulation of expression depends principally on the longterminal repeat (LTR) which contains signals for enhancement, promotion,initiation and polyadenylation of RNA synthesis. Transcription isinitiated at the left end of the R sequences in the left-hand LTR. Viraltranscription is catalyzed by a cellular type II RNA polymerase and ispolyadenylated post-transcriptionally near the right hand end of theright LTR. The U3 regions of all LTRs possess CCAAT and TATAA boxes.Enhancer function has been localized to a repeat sequence of variablelength (72 to 101 nucleotides) that is found in U3, upstream from thepromoter domain. Sequences in U3 of the Moloney murine leukemia virus(MMTV) LTR have also been found to be responsible for the enhanced levelof virus expression in response to glucocorticoid treatment ofMMTV-infected cells.

Murine parvovirus (MVM) which displays a natural tropism for human Tcells, may be modified to infect hepatocytes or the endothelial cells ofthe gut. In the case of MVM, the major cis-acting factors that areessential for the first steps in viral DNA replication are foundupstream of the “early” promoter, P4 (Cotmore et.al., 1992, Virology190:365-377). The early promoter is the initiating promoter for viraltranscription, and its TATAA box is located 4 map units from the leftend of the MVM genome, at around nucleotide 150. The essentialcis-acting functions for the origin of MVM DNA replication are alsofound in this region. The insertion of foreign DNA sequences such as thecoding sequences for the ApoA-1 peptides just downstream of this regionplaces them under the regulation of viral gene expression withoutdisruption of other essential viral activities, i.e., DNA replication.

Vesicular stomatitis virus (VSV), the prototypic rhabdovirus, can beused in gene therapy to express the ApoA-I peptides of the invention inmammalian cells. VSV is the simplest of enveloped animal viruses, growsto very high titers and can be prepared in large quantities. Foreignglycoproteins may be incorporated into the VSV G envelope protein totarget the viral vector to liver cells or cells of the small and largeintestine. The VSV G gene is large enough to accommodate nucleotidesequences encoding the ApoA-I peptides of the invention. (See Schnell etal., 1996, Proc. Natl. Acad. Sci. USA 93:11359-11365).

Alphavirus-based expression vectors, a prototype of envelopedpositive-strand RNA viruses, can also be engineered to mediate efficientexpression of the nucleotides encoding the ApoA-I peptides of theinvention. Although normally cytocidal for vertebrate cells, variants ofalphavirus with adaptive mutations allow noncytopathic replication ofalphaviral vectors. Nucleotide sequences encoding the ApoA-I peptidesmay be engineered into the structural gene of the replication defectivealphavirus genome. Defective helper RNAs containing the cis-actingsequences required for replication as well as the RNA promoter topackage the replication defective alphaviral vector (Frolov et al.,1996, Proc. Natl. Acad. Sci. USA 93:11371-11377). In addition to VSV andalphavirus and viruses, including influenza, rhababviruses,parainfluenza virus and bunya virus, may also be engineered usingsimilar techniques for the delivery and expression of the nucleotidesequences encoding the ApoA-I peptides of the present invention.

The viral vectors described above are by way of example, and not by wayof limitation of the present invention. Any viral vector which may begenetically engineered to safely administer the nucleotide sequencesencoding ApoA-I peptides and agonists to the target cells may be used inaccordance with the present invention.

5.2.2. HOST CELLS EXPRESSING APoA-I

The present invention encompasses the expression of ApoA-I and peptidesexhibiting ApoA-1 activity as described above, in animal cells or celllines, preferably human cell or cell lines which may then beadministered in vivo. The host cells which may be used in accordancewith the invention to express ApoA-I and peptides exhibiting ApoA-Iactivity, including, but are not limited to, fibroblasts, Caco-2 cells,epithelial cells, endothelial cells, muscle cells, hepatocytes, cellsisolated from large and small intestine etc. In a preferred embodimentof the invention, hepatocytes and cells isolated from the smallintestine are used as host cells to express ApoA-I and peptidesexhibiting ApoA-I activity. The host cells of the present invention alsohave utility as a model system to further understand the role of ApoAIin lipid metabolism.

The host cells of the present invention also have utility as a modelsystem to further understand the role of ApoA-I in lipid metabolism.

In one embodiment of the present invention, host cells are obtained fromthe recipient, that is the individual who is to receive the transducedcells, or from a donor. Examples of cells which may be geneticallyengineered in accordance with the present invention, include, but arenot limited to, hepatocytes, gall bladder cells, cells of the smallintestine, epithelial cells, and endothelial cells, including cellsisolated from small and large blood vessels. The isolate cells may betransfected or transduced with the DNA and viral vectors describedherein. The transduced cells may subsequently be grafted or transplantedinto the recipient.

In another embodiment of the present invention, both transient andpermanent recombinant cell lines may be engineered in which the nativeapolipoprotein A, or any other apolipoprotein, coding sequence isreplaced by the coding sequence for a modified preproapolipoprotein A-I,a modified proapolipoprotein A-I, a modified ApoA-I or an Apo A-Iagonist.

A host cell strain may be chosen which modulates the expression of theinserted sequences, or modifies and processes the gene product in thespecific fashion desired. Such modification (e.g., glycosylation) andprocessing (e.g. cleavage) of protein products may be important for thefunction of the protein. Different host cells have characteristic andspecific mechanisms for the post-translational processing andmodification of proteins and gene products. Appropriate cell lines orhost systems can be chosen to ensure the correct modification of theforeign protein expressed. To this end, eukaryotic host cells whichpossess the cellular machinery for proper processing of the primarytranscript, glycosylation, and phosphorylation of the gene product maybe used. Such mammalian host cells include but are not limited to CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3 and WI38 cell lines.

For long term, high-yield production of native ApoA-I or peptides havingApoA-I agonist activity mammalian host cells stably expressing thesepeptides may be engineered. Rather than using expression vectors whichcontain viral origins of replication, host cells can be transformed withDNA controlled by appropriate expression control elements (e.g.,promoter, enhancer, sequences, transcription terminators,polyadenylation sites, etc.), and a selectable marker. Following theintroduction of the foreign DNA, engineered cells may be allowed to growfor 1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method may advantageouslybe used to engineer cell lines. This method may advantageously be usedto engineer cell lines which express the ApoA-I and ApoA-I peptide geneproducts. Such cell lines would be particularly useful in screening andevaluation of compounds that affect the endogenous activity of theApoA-I and ApoA-I peptide gene product.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigler,et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981, Proc.Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Nati. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

For example, the cassettes encoding heteropolymers of ApoA-I peptidesmay be advantageously engineered into adenoviruses as described indetail above.

5.2.3. TRANSGENIC ANIMALS EXPRESSING APOA-I

The invention also encompasses the expression of the ApoA-I nucleotidesequences of the present invention transgenic animals as a model systemto further understand the role of ApoA-I in lipid metabolism in additionto having utility for gene therapy. Animals of any species, including,but not limited to, mice, rats, rabbits, guinea pigs, micro-pigs, goats,and non-human primates, e.g., baboons, monkeys, and chimpanzees may beused to generate ApoA-I transgenic animals.

Any technique known in the art may be used to introduce the ApoA-Itransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P.C. and Wagner, T.E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry theApoA-I transgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. Thetransgene may be integrated as a single transgene or in concatamers,e.g., head-to-head tandems or head-to-tail tandems. The transgene mayalso be selectively introduced into and activated in a particular celltype by following, for example, the teaching of Lasko et al. (Lasko, M.et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236). The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell type of interest, and will be apparent to thoseof skill in the art. When it is desired that the ApoA-I transgene beintegrated into the chromosomal site of the endogenous ApoA-I gene, genetargeting is preferred. Briefly, when such a technique is to beutilized, vectors containing some nucleotide sequences homologous to theendogenous ApoA-I gene are designed for the purpose of integrating, viahomologous recombination with chromosomal sequences, into and disruptingthe function of the nucleotide. sequence of the endogenous ApoA-I gene.The transgene may also be selectively introduced into a particular celltype, thus inactivating the endogenous ApoA-I gene in only that celltype, by following, for example, the teaching of Gu et al. (Gu, et al.,1994, Science 265: 103-106). The regulatory sequences required for sucha cell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art.

5.3. GENE THERAPY APPROACHES TO DELIVER ApoA-I PEPTIDES

Gene therapy approaches may also be used in accordance with the presentinvention to deliver nucleotide sequences encoding native ApoA-I orApoA-I peptides.

The gene therapy approaches of the present invention can be used totreat any disorder in animals, especially mammals, including humans, forwhich increasing serum HDL concentration, activating LCAT, and promotingcholesterol efflux and RCT is beneficial. Such conditions include, butare not limited to hyperlipidemia, and especially hypercholesteremia,and cardiovascular disease such as atherosclerosis (including preventionof atherosclerosis and treatment of existing disease); restenosis (e.g.,preventing or treating atherosclerotic plaques which develop as aconsequence of medical procedures such as balloon angioplasty); andother disorders, such as endotoxemia which often results in septic shock(e.g., Gouni et al., 1993, J. Lipid Research 94:139-146; Levine,WO96/04914).

The gene therapy approaches of the present invention can be used aloneor in combination therapy with other drugs used to treat the foregoingconditions. Such therapies include, but are not limited to simultaneousor sequential administration of the drugs involved.

5.3.1. GENE REPLACEMENT THERAPY

With respect to an increase in the level of normal ApoA-I peptideexpression and/or ApoA-I peptide product activity, ApoA-I peptidenucleic acid sequences, described, above, in Section 5.1, can, forexample, be utilized for the treatment of dislipidemic or hyperlipidemicdisorders Such treatment can be administered, for example, in the formof gene replacement therapy. Specifically, one or more copies of anormal ApoA-I peptide or a portion of the ApoA-I peptide that directsthe production of a ApoA-I peptide product exhibiting normal ApoA-Ipeptide function, may be inserted into the appropriate cells within apatient, using vectors that include, but are not limited to adenovirus,adeno-associated virus, and retrovirus vectors, in addition to otherparticles that introduce DNA into cells, such as liposomes.

Because the ApoA-I protein is expressed in the liver, such genereplacement therapy techniques should be capable delivering ApoA-Ipeptide sequences to these cell types within patients.

In another embodiment, techniques for delivery involve directadministration of such ApoA-I peptide sequences to the site of the cellsin which the ApoA-I peptide sequences are to be expressed.

Additional methods that may be utilized to increase the overall level ofApoA-I peptide expression and/or ApoA-I peptide product activity includethe introduction of appropriate ApoA-I expressing cells, preferablyautologous cells, into a patient at positions and in numbers that aresufficient to ameliorate the symptoms of a hyperlipidemia disorder. Suchcells may be either recombinant or non-recombinant.

Among the cells that can be administered to increase the overall levelof ApoA-I peptide expression in a patient are normal cells, preferablyhepatocytes, that express the ApoA-I peptide.

Alternatively, cells, preferably autologous cells, can be engineered toexpress ApoA-I peptide sequences, and may then be introduced into apatient in positions appropriate for the amelioration of the symptoms ofa dyslipidemia disorder. Alternately, cells that express an unimpairedApoA-I peptide and that are from a MHC matched individual can beutilized, and may include, for example, hepatocytes. The expression ofthe ApoA-I peptide sequences is controlled by the appropriate generegulatory sequences to allow such expression in the necessary celltypes. Such gene regulatory sequences are well known to the skilledartisan. Such cell-based gene therapy techniques are well known to thoseskilled in the art, see, e.g., Anderson, U.S. Pat. No. 5,399,349incorporated herein by reference in its entirety.

When the cells to be administered are non-autologous cells, they can beadministered using well known techniques that prevent a host immuneresponse against the introduced cells from developing. For example, thecells may be introduced in an encapsulated form which, while allowingfor an exchange of components with the immediate extracellularenvironment, does not allow the introduced cells to be recognized by thehost immune system.

5.3.2. DELIVERY OF NUCLEIC ACIDS IN VIVO

Delivery of the nucleic acid into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient for cell replacement therapy. These two approaches areknown, respectively, as in vivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (see U.S. Pat. No. 4,980,286), or by direct injection of nakedDNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, or by administering it in linkage to a peptide which isknown to enter the cell or nucleus, e.g., by administering it in linkageto a ligand subject to receptor-mediated endocytosis (see e.g., Wu andWu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to targetcell types specifically expressing the receptors), etc. In a specificembodiment, the nucleic acid can be targeted in vivo for cell specificuptake and expression, by targeting a specific receptor (see, e.g., PCTPublications WO 92/06180 dated Apr. 16, 1992 (Wu et al.); WO 92/22635dated Dec. 23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, 1992(Findeis et al.); WO93/14188 dated Jul. 22, 1993 (Clarke et al.), WO93/20221 dated Oct. 14, 1993 (Young)). In another embodiment, a nucleicacid-ligand complex can be formed in which the ligand comprises afusogenic viral peptide to disrupt endosomes, allowing the nucleic acidto avoid lysosomal degradation. Alternatively, the nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination (Koller & Smithies, 1989, Proc.Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al., 1989, Nature342:435-438).

In yet another embodiment, the nucleic acid may be administered in vivoby a time controlled release device. The nucleic acid may also beadministered in vivo by a device which contains cells expressing theApoA-I peptides and agonists of the present invention, protecting themfrom rejection by the recipient or host. In accordance with this device,the proteins and peptides may diffuse through a permeable membrane whichhas a molecular weight cut off of approximately 3,500 to 50,000 daltons.

One strategy for targeting DNA or viral vectors hepatocytes can be basedon the presence of the asialoglycoprotein (ASPG) receptor onhepatocytes. This receptor is specifically expressed on the cell surfaceof hepatocytes. Asialoglycopeptide-protein conjugates andasialoglycopeptide-coated vesicles have been used to specificallydeliver to a variety of bioactive agents to the liver in vivo. Aitie etal., 1980, Proc. Natl. Acad. Sci. 27:5923-5927; Hildenbrandt et al.,1980, BBA 631:499-502.

Another strategy to targeting delivery of nucleotide sequences tohepatocytes is to genetically modify viral vectors, i.e. adenovirusesand retroviruses to express ligands for the ASPG receptors.Internalization of virus occurs through the specific interaction of theviral envelope with a cell surface receptor, followed byreceptor-mediated endocytosis of the virus/receptor complex. The viralvectors which may be used in accordance with the present invention arediscussed above in Section 5.2.2.

5.3.3. DELIVERY OF NUCLEIC ACIDS EX VIVO

Another approach to gene therapy, for use in the cell replacementtherapy of the invention, involves transferring a gene to cells intissue culture by such methods as electroporation, lipofection, calciumphosphate mediated transfection, or viral infection. Usually, the methodof transfer includes the transfer of a selectable marker to the cells.The cells are then placed under selection to isolate those cells thathave taken up and are expressing the transferred gene. Those cells arethen delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including, but not limited to, transfection, electroporation,microinjection, infection with a viral vector containing the nucleicacid sequences, cell fusion, chromosome-mediated gene transfer,microcell-mediated gene transfer, spheroplast fusion, etc. Numeroustechniques are known in the art for the introduction of foreign genesinto cells (see e.g., Loeffler & Behr, 1993, Meth. Enzymol. 217:599-618;Cohen et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac.Ther. 29:69-92) and may be used in accordance with the presentinvention, provided that the necessary developmental and physiologicalfunctions of the recipient cells are not disrupted. The technique shouldprovide for the stable transfer of the nucleic acid to the cell, so thatthe nucleic acid is expressible by the cell and preferably heritable andexpressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. In a preferred embodiment, epithelial cellsare injected, e.g., subcutaneously. In another embodiment, recombinantskin cells (e.g., keratinocytes) may be applied as a skin graft onto thepatient. Recombinant blood cells (e.g., hematopoietic stem or progenitorcells) are preferably administered intravenously. The amount of cellsenvisioned for use depends on the desired effect, patient state, etc.,and can be determined by one skilled in the art.

In an embodiment in which recombinant cells are used in gene therapy,nucleotides which encode a gene or promoter suppressor are introducedinto the cells such that it is expressible by the cells or theirprogeny, and the recombinant cells are then administered in vivo fortherapeutic effect. In a specific embodiment, stem or progenitor cellsare used. Any stem and/or progenitor cells which can be isolated andmaintained in vitro can potentially be used in accordance with thisembodiment of the present invention.

By way of example, and not by limitation, for ex vivo gene therapyapproaches, the following procedures may be utilized for the isolationof hepatocytes to be genetically engineered to express the ApoA-Ipeptides of the present invention and the grafting or transplantation ofthe engineered hepatocytes into the recipient.

In this embodiment, hepatocytes are obtained from a donor or arecipient, the individual who is to receive the engineered hepatocytes.This procedure entails removing a portion of a liver, from whichhepatocytes are removed by in situ perfusion of a collagenase solution.If the hepatocytes are to be isolated from an intact liver, a catheteris inserted into a vein which either leaves or enters the liver,collagenase solution is perfused through the catheterized vessels,resulting in release of hepatocytes. Once removed or isolated, thehepatocytes are plated and maintained under conditions suitable fortransfection.

For example, several methods have been described for isolating highlyenriched populations of rat hepatocytes and maintaining these cells inculture for extended periods of time. Koch, K. S. and H. L. Leffert,Annals N.Y. Academy of Sciences, 349:111-127 (1980); McGowan, J. A. etal., Journal of Cellular Physiology, 108:353-363 (1981); Bissell, D. M.and P. S. Guzelian, Annals of the New York Academy of Sciences,349:85-98 (1981); and Enat, R. et al., Proceedings of the NationalAcademy of Sciences, U.S.A., 81:1411-1415 (1984). Such methods can beused to isolate and maintain hepatocytes to be transduced by the methodof the present invention. Hepatocytes can also be prepared using amodification of the procedure developed by Barry and Friend, describedbelow and in Example 1, with the perfusion mixture described by Leffert.Leffert, H. L. et al., Methods in Enzymology, 58:536-544 (1979), theteachings of which are incorporated herein by reference.

The genetic material incorporated into and expressed by hepatocytes canalso, optionally, include genetic material encoding a selectable marker,thus making it possible to identify and select cells which contain andexpress the genetic material of interest.

Thus, DNA or RNA introduced into cultured hepatocytes of the presentinvention includes the genetic material (DNA or RNA) of interest and,optionally, genetic material encoding a selectable marker; such DNA orRNA is referred to as incorporated genetic material (or incorporatedDNA, incorporated RNA). Hepatocytes containing incorporated geneticmaterial are referred to as transduced hepatocytes; they express the DNAor RNA of interest and produce the encoded protein or polypeptide.

Exogenous DNA encoding a polypeptide or protein of interest and,optionally, a selectable marker (e.g., neo, which encodes neomycinresistance) is incorporated in vitro into hepatocytes as described belowand in Examples I-III. Hepatocytes isolated as described previously areplated at subconfluent density on matrix substrata and maintained inhormonally defined media, such as that described by Enat et al., theteachings of which are incorporated herein by reference. Enat, R., etal., Proceedings of the National Academy of Sciences, USA, 81:1411-1415(1984). The media is changed as needed to maintain the hepatocytes.

Cells are subsequently infected with an amphotropic retrovirus whichcontains DNA of interest (e.g., DNA encoding a polypeptide whoseexpression in hepatocytes is desired) and, optionally, DNA encoding aselectable marker to be incorporated into the hepatocytes. Thehepatocytes are infected with the recombinant adenovirus or retrovirus(and thus transduced with the DNA of interest) by exposing them to viruswhich has a recombinant genome. This results in infection of the cellsby the recombinant retrovirus. It is possible to optimize the conditionsfor infection of the hepatocytes by using a high titer amphotropicvirus.

Viral stocks, to be used in introducing genetic material of interestinto hepatocytes, are harvested, as described above, supplemented withPolybrene (Aldrich) and added to the culture of hepatocytes. If thetiter of the virus is high (e.g., approximately 10⁶ cfu per ml.), thenvirtually all hepatocytes will be infected and no selection oftransduced he patocytes is required. If the titer is very low, then itis necessary to use a retroviral vector that has a selectable marker,such as neo or his. If a selectable marker is used, after exposure tothe virus, the cells are grown to confluence and split into selectivemedia (e.g., media containing G418 if the selectable marker is neo,media containing histidinol and no histidine if the selectable marker ishis).

Hepatocytes expressing the incorporated genetic material are grown toconfluence in tissue culture vessels; removed from the culture vessel;and introduced into the body. This can be done surgically, for example.In this case, the tissue which is made up of transduced hepatocytescapable of expressing the nucleotide sequence of interest is grafted ortransplanted into the body. For example, it can be placed in theabdominal cavity in contact with/grafted onto the liver or in closeproximity to the liver. Alternatively, the transducedhepatocyte-containing tissue can be attached to microcarrier beads,which are introduced (e.g., by injection) into the peritoneal space ofthe recipient. This approach has been shown to be successful bytransplantation of wild type hepatocytes into a strain of rats (Nagaseanalbuminemic rats) which are deficient in albumin synthesis anddemonstration of moderate levels of albumin in serum of transplantedanimals. Direct injection of genetically modified hepatocytes into theliver may also be possible.

Once introduced into the body of an individual, the transducedhepatocytes provide a continuous supply of the hormone, enzyme or drugencoded by the genetic material of interest. The amount of the hormone,enzyme or drug supplied in this way can be modified or regulated asneeded (e.g., by using external cues or factors which control or affectproduction, by controlling the size of the graft or the quantity offibroblasts introduced into the body, or by removing the graft).

5.4. PHARMACEUTICAL FORMULATIONS AND METHODS OF ADMINISTRATION

The present invention encompasses pharmaceutical formulations for thedelivery of DNA or viral vectors encoding ApoA-I or ApoA-I peptides forin vivo transformation of cells or the delivery of host cellstransformed with DNA or viral vectors for in vivo and ex vivo genetherapy approaches.

The ApoA-I agonists of the invention can be used to treat any disorderin animals, especially mammals including humans, for which increasingserum HDL concentration, activating LCAT, and promoting cholesterolefflux and RCT is beneficial. Such conditions include, but are notlimited to hyperlipidemia, and especially hypercholesteremia, andcardiovascular disease such as atherosclerosis (including prevention ofatherosclerosis and treatment of existing disease); restenosis (e.g.,preventing or treating atherosclerotic plaques which develop as aconsequence of medical procedures such as balloon angioplasty); andother disorders, such as endotoxin-induced shock which often results inhypertriglyceridemia (see, e.g., Gouni et al., 1993, J. Lipid Research94:139-146; Levine, WO96/04914).

The ApoA-I agonists can be used alone or in combination therapy withother drugs used to treat the foregoing conditions. Such therapiesinclude, but are not limited to simultaneous or sequentialadministration of the drugs involved.

For example, in the treatment of hypercholesterolemia oratherosclerosis, mammalian host cells expressing the ApoA-I agonistformulations can be administered with any one or more of the cholesterollowering therapies currently in use; e.g., bile-acid resins, niacin,and/or statins. Such a combined regimen may produce particularlybeneficial therapeutic effects since each drug acts on a differenttarget in cholesterol synthesis and transport; i.e., bile-acid resinsaffect cholesterol recycling, the chylomicron and LDL population; niacinprimarily affects the VLDL and LDL population; the statins inhibitcholesterol synthesis, decreasing the LDL population (and perhapsincreasing LDL receptor expression); whereas the ApoA-I agonists affectRCT, increase HDL, increase LCAT activity and promote cholesterolefflux.

In another embodiment, mammalian host cells expressing ApoA-I agonistsmay be used in conjunction with fibrate to treat hyperlipidemia,hypercholesterolemia and/or cardiovascular disease such asatherosclerosis. This regimen may likewise prove very beneficial sincefibrate does not have a proven effect on cardiovascular disease.

In yet another embodiment, the ApoA-I agonists of the invention can beused in combination with the anti-microbials and anti-inflammatoryagents currently used to treat septic shock induced by endotoxin.

The ApoA-I agonists expressed by the nucleotide sequences of theinvention can be formulated as peptides or as peptide-lipid complexeswhich can be administered to subjects in a variety of ways to deliverthe ApoA-l agonist to the circulation. Exemplary formulations andtreatment regimens are described below.

The mammalian host cells expressing ApoA-I peptide agonists, theDNA-lipid complexes or “naked” plasmid DNA encoding the ApoA-I agonistsof the invention may be administered by any suitable route that ensuresbioavailability in the circulation. This can best be achieved byparenteral routes of administration, including intravenous (IV),intramuscular (IM), intradermal, subcutaneous (SC) and intraperitoneal(IP) injections. However, other routes of administration may be used.For example, absorption through the gastrointestinal tract can beaccomplished by oral routes of administration (including but not limitedto ingestion, buccal and sublingual routes) provided appropriateformulations (e.g., enteric coatings) are used to avoid or minimizedegradation of the active ingredient, e.g., in the harsh environments ofthe oral mucosa, stomach and/or small intestine. Furthermore, one mayadminister the agent of the present invention in a targeted drugdelivery system, for example in a liposome targeted to the liver. Theliposomes will be targeted to an taken up selectively by liver cells.Alternatively, administration via mucosal tissue such as vaginal andrectal modes of administration may be utilized to avoid or minimizedegradation in the gastrointestinal tract. In yet another alternative,the formulations of the invention can be administered transcutaneously(e.g., transdermally), or by inhalation. It will be appreciated that thepreferred route may vary with the condition, age and compliance of therecipient.

The actual dose of ApoA-I agonists or peptide-lipid complex used willvary with the route of administration, and should be adjusted to achievecirculating plasma concentrations of 100 mg to 2 g/l. Data obtained inanimal model systems described herein show that the ApoA-I agonists ofthe invention associate with the HDL component, and have a projectedhalf-life in humans of about five days. Thus, in one embodiment, theApoA-I agonists can be administered by injection at a dose between 0.5to 100 mg/kg (dose/IV;IM;SC) once a week. In another embodiment,desirable serum levels may be maintained by continuous infusion toprovide about 0.5-100 mg/kg/hr or by intermittent infusion providingabout 0.5-100 mg/kg.

Toxicity and therapeutic efficacy of the various ApoA-I agonists can bedetermined using standard pharmaceutical procedures in cell culture orexperimental animals for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀ (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.ApoA-I peptide agonists which exhibit large therapeutic indices arepreferred.

5.4.1. PHARMACEUTICAL FORMULATIONS

The pharmaceutical formulation of the invention contain the mammalianhost cells expressing the ApoA-I peptide agonist, the DNA encoding theApoA-I peptide either naked or complexed with lipids, liposomes, ornonlipid cationic polymers as the active ingredient in apharmaceutically acceptable carrier suitable for administration anddelivery in vivo.

Injectable preparations include sterile suspensions, solutions oremulsions of the active ingredient in aqueous or oily vehicles. Thecompositions may also contain formulating agents, such as suspending,stabilizing and/or dispersing agent. The formulations for injection maybe presented in unit dosage form, e.g., in ampules or in multidosecontainers, and may contain added preservatives.

Alternatively, the injectable formulation may be provided in powder formfor reconstitution with a suitable vehicle, including but not limited tosterile pyrogen free water, buffer, dextrose solution, etc., before use.To this end, the ApoA-I agonist peptide may be lyophilized, or theco-lyophilized peptide-lipid complex may be prepared. The storedpreparations can be supplied in unit dosage forms and reconstitutedprior to use in vivo.

For prolonged delivery, the active ingredient can be formulated as adepot preparation, for administration by implantation; e.g.,subcutaneous, intradermal, or intramuscular injection. Thus, forexample, the active ingredient may be formulated with suitable polymericor hydrophobic materials (e.g., as an emulsion in an acceptable oil) orion exchange resins, or as sparingly soluble derivatives; e.g., as asparingly soluble salt form of the ApoA-I agonist.

Alternatively, transdermal delivery systems manufactured as an adhesivedisc or patch which slowly releases the active ingredient forpercutaneous absorption may be used. To this end, permeation enhancersmay be used to facilitate transdermal penetration of the activeingredient. A particular benefit may be achieved by incorporating theApoA-I agonists of the invention or the peptide-lipid complex into anitroglycerin patch for use in patients with ischemic heart disease andhypercholesterolemia.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated to give controlled release ofthe active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner. For rectal and vaginalroutes of administration, the active ingredient may be formulated assolutions (for retention enemas) suppositories or ointments.

For administration by inhalation, the active ingredient can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g. gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

5.5. OTHER USES

The ApoA-I peptides and agonists encoded by the nucleotide sequences ofthe invention can be used in assays in vitro to measure serum HDL, e.g.,for diagnostic purposes. Because the ApoA-I agonists associate with theHDL component of serum, the agonists can be used as “markers” for theHDL population. Moreover, the agonists can be used as markers for thesubpopulation of HDL that are effective in RCT. To this end, the agonistcan be added to or mixed with a patient serum sample; after anappropriate incubation time, the HDL component can be assayed bydetecting the incorporated ApoA-I agonist. This can be accomplishedusing labeled agonist (e.g., radiolabels, fluorescent labels, enzymelabels, dyes, etc.), or by immunoassays using antibodies (or antibodyfragments) specific for the agonist.

Alternatively, labeled agonist can be used in imaging procedures (e.g.,CAT scans, MRI scans) to visualize the circulatory system, or to monitorRCT, or to visualize accumulation of HDL at fatty streaks,atherosclerotic lesions, etc. (where the HDL should be active incholesterol efflux).

6. EXAMPLE: LCAT ACTIVATION ASSAY

Examples of peptides which may be encoded by the nucleotides of thepresent invention are listed in Table VIII which also includes peptideswhich are chemically synthesized. Only those peptides composed of aminoacids which may be genetically encoded or which exhibit an LCAT activityof less than 38% as compared with native ApoA-I are encompassed by thepresent invention. All of the peptides listed were analyzed in vitro fortheir ability to activate LCAT. In the LCAT assay, substrate vesicles(small unilamellar vesicles or “SUVs”) composed of eggphophatidylcholine or 1-Palmitoyl-2-oleyl-phosphatidyl-choline (POPC)and radiolabelled cholesterol are preincubated with equivalent masseseither of peptide or ApoA-I (isolated from human plasma). The reactionis initiated by addition of LCAT (purified from human plasma). NativeApoA-I, which was used as positive control, represents 100% activationactivity. “Specific activity” (i.e., units of activity (LCATactivation)/unit of mass) of the peptides can be calculated as theconcentration of peptide that achieves maximum LCAT activation. Forexample, a series of concentrations of the peptide (e.g., a limitingdilution) can be assayed to determine the “specific activity” for thepeptide—the concentration which achieves maximal LCAT activation (i.e.,percentage conversion of cholesterol to cholesterol ester) at a specifictimepoint in the assay (e.a., 1 hr.). When plotting percentageconversion of cholesterol at, e.g., 1 hr., against the concentration ofpeptide used, the “specific activity” can be identified as theconcentration of peptide that achieves a plateau on the plottal curve.

6.1. PREPARATION OF SUBSTRATE VESICLES

The vesicles used in the LCAT assay are SUVs composed of eggphosphatidylcholine (EPC) or 1-palmitoyl-2-oleyl-phosphatidylcholine(POPC) and cholesterol with a molar ratio of 20:1. To prepare a vesiclestock solution sufficient for 40 assays, 7.7 mg EPC (or 7.6 mg POPC; 10μmol), 78 μg (0.2 μmol) 4-¹⁴C-cholesterol, 116 μg cholesterol (0.3 μmol)are dissolved in 5 ml xylene and lyophilized. Thereafter 4 ml of assaybuffer is added to the dry powder and sonicated under nitrogenatmosphere at 4° C. Sonication conditions: Branson 250 sonicator, 10 mmtip, 6.5 minutes; Assay buffer: 10 mM Tris, 0.14M NaCl, 1 mM EDTA, pH7.4). The sonicated mixture is centrifuged 6 times for 5 minutes eachtime at 14,000 rpm (16,000×g) to remove titanium particles. Theresulting clear solution is used for the enzyme assay.

6.2. PURIFICATION OF LCAT

For the LCAT purification, dextran sulfate/Mg²⁺ treatment of humanplasma is used to obtain lipoprotein deficient serum (LPDS), which issequentially chromatographed on Phenylsepharose, Affigelblue,ConcanavalinA sepharose and anti-ApoA-I affinity chromatography, assummarized for a representative purification in Table VII, below: TABLEVII LCAT PURIFICATION Total Total Total Volume Protein Activity YieldPurification Fraction (mL) (mg) (nmol CE/mg * h) (%) (fold) Plasma 55044550 63706 LPDS 500 31000 62620 98 1.4 Phenyl 210 363 51909 82 100sepharose Affigel  95 153 25092 39 115 blue ConA  43 36 11245 18 220sepharose Anti-A-I 120 3.5  5500  9 1109 Affinity

6.2.1. PREPARATION OF LPDS

To prepare LPDS, 500 ml plasma is added to 50 ml dextran sulfate(MW=500000) solution. Stir 20 minutes. Centrifuge for 30 minutes at 3000rpm (16,000×g) at 4° C. Use supernatant (LPDS) for further purification(ca. 500 ml).

6.2.2. PHENYLSEPHAROSE CHROMATOGRAPHY

The following materials and conditions were used for the phenylsepharosechromatography. solid phase: Phenylsepharose fast flow, high subst.grade, Pharmacia column: XK26/40, gel bed height: 33 cm, V = ca. 175 mlflow rates: 200 ml/hr (sample) wash: 200 ml/hr (buffer) elution:  80ml/hr (distilled water) buffer:  10 mM Tris, 140 mM NaCl, 1 mM EDTApH7.4, 0.01% Na-azide.

Equilibrate the column in Tris-buffer, add 29 g NaCl to 500 ml LPDS andapply to the column. Wash with several volumes of Tris buffer until theabsorption at 280 nm wavelength is approximately at the baseline, thenstart the elution with distilled water. The fractions containing proteinare pooled (pool size: 180 ml) and used for Affigelblue chromatography.

6.2.3. AFFIGELBLUE CHROMATOGRAPHY

The Phenylsepharose pool is dialyzed overnight at 4° C. against 20 mMTris-HCl, pH7.4, 0.01% Na-azide. The pool volume is reduced byultrafiltration (Amicon YM30) to 50-60 ml and loaded on an Affigelbluecolumn. solid phase: Affigelblue, Biorad, 153-7301 column, XK26/20, gelbed height: ca. 13 cm; column volume: approx. 70 ml. flow rates:loading: 15 ml/h wash: 50 ml/hEquilibrate column in Tris-buffer. Apply Phenylsepharose pool to column.Start in parallel to collect fractions. Wash with Tris-buffer. Thepooled fractions (170 ml) were used for ConA chromatography.

6.2.4. ConA CHROMATOGRAPHY

The Affigelblue pool was reduced via Amicon (YM30) to 30-40 ml anddialyzed against ConA starting buffer (1 mM Tris HCl pH7.4; 1 mM MgCl₂,1 mM MnCl₂, 1 mM CaCl₂, 0.01% Na-azide) overnight at 4° C. solid phase:ConA sepharose (Pharmacia) column: XK26/20, gel bed height: 14 cm (75ml) flow rates: loading 40 ml/h washing (with starting buffer): 90 ml/helution: 50 ml/h, 0.2 M Methyl-α-D-mannoside in 1 mM Tris, pH 7.4.The protein fractions of the mannoside elutions were collected (110 ml),and the volume was reduced by ultrafiltration (YM30) to 44 ml. The ConApool was divided in 2 ml aliquots, which are stored at −20° C.

6.2.5. ANTI-APoA-I AFFINITY CHROMATOGRAPHY

Anti-ApoA-I affinity chromatography was performed on Affigel-Hz material(Biorad), to which the anti-ApoA-I abs have been coupled covalently.column: XK16/20, V = 16 ml. The column was equilibrated with PBS pH 7.4.Two ml of the ConA pool was dialyzed for 2 hours against PBS beforeloading onto the column. flow rates: loading: 15 ml/hour washing (PBS)40 ml/hour.The pooled protein fractions (V=14 ml) are used for LCAT assays.

The column is regenerated with 0.1 M. Citrate buffer (pH 4.5) to elutebound A-I (100 ml), and immediately after this procedure reequilibratedwith PBS.

6.3. RESULTS

The results of the LCAT activation assay are presented in Table VIII,infra. TABLE VIII LCAT ACTIVATION EXHIBITED BY EXEMPLARY CORE PEPTIDESACTIVITY He (%) He (%) He (%) He (%) PEPTIDE AMINO ACID SEQUENCE (%)LCAT free mics SUVs TFE  1 (SEQ ID NO: 1) PVLDLFRELLNELLEZLKQKLK 120% 77 85 81 69  2 (SEQ ID NO: 2) GVLDLFRELLNELLEALKQKLKK 105%  3 (SEQ IDNO: 3) PVLDLFRELLNELLEWLKQKLK 98%  70 95 80 95  4 (SEQ ID NO: 4)PVLDLFRELLNELLEALKQKLK 93%  80 95 97 94  5 (SEQ ID NO: 5)pVLDLFRELLNELLEALKQKLKK 90%  6 (SEQ ID NO: 6) PVLDLFRELLNEXLEALKQKLK 80% 57 93 70 99  7 (SEQ ID NO: 7) PVLDLFKELLNELLEALKQKLK 83%  77 89 85 73 8 (SEQ ID NO: 8) PVLDLFRELLNEGLEALKQKLK 83%  20 90 61 93  9 (SEQ ID NO:9) PVLDLFRELGNELLEALKQKLK 83%  10 (SEQ ID NO: 10) PVLDLFRELLNELLEAZKQKLK79%  60 87 70 71  11 (SEQ ID NO: 11) PVLDLFKELLQELLEALKQKLK 72%  12 (SEQID NO: 12) PVLDLFRELLNELLEAGKQKLK 70%  13 (SEQ ID NO: 13)GVLDLFRELLNEGLEALKQKLK 67%  14 (SEQ ID NO: 14) PVLDLFRELLNELLEALOQOLO61%  70 96 80  15 (SEQ ID NO: 15) PVLDLFRELWNELLEALKQKLK 60%  55 60 6468  16 (SEQ ID NO: 16) PVLDLLRELLNELLEALKQKLK 59%  17 (SEQ ID NO: 17)PVLELFKELLQELLEALKQKLK 59%  18 (SEQ ID NO: 18) GVLDLFRELLNELLEALKQKLK58%  19 (SEQ ID NO: 19) pVLDLFRELLNEGLEALKQKLK 58%  20 (SEQ ID NO: 20)PVLDLFREGLNELLEALKQKLK 57%  21 (SEQ ID NO: 21) pVLDLFRELLNELLEALKQKLK57%  22 (SEQ ID NO: 22) PVLDLFRELLNELLEGLKQKLK 54%  23 (SEQ ID NO: 23)PLLELFKELLQELLEALKQKLK 54%  24 (SEQ ID NO: 24) PVLDLFRELLNELLEALQKKLK53%  25 (SEQ ID NO: 25) PVLDFFRELLNEXLEALKQKLK 51%  46 82 93  26 (SEQ IDNO: 26) PVLDLFRELLNELLELLKQKLK 47%  27 (SEQ ID NO: 27)PVLDLFRELLNELZEALKQKLK 44%  72 92 82 81  28 (SEQ ID NO: 28)PVLDLFRELLNELWEALKQKLK 40%  82 98 90 81  29 (SEQ ID NO: 29)AVLDLFRELLNELLEALKQKLK 39%  30 (SEQ ID NO: 30) PVLDLFRELLNELLEALKQKLK¹38%  85 90 98 90  31 (SEQ ID NO: 31) PVLDLFLELLNEXLEALKQKLK 34%  49 9890  32 (SEQ ID NO: 32) XVLDLFRELLNELLEALKQKLK 33%  33 (SEQ ID NO: 33)PVLDLFREKLNELLEALKQKLK 33%  34 (SEQ ID NO: 34) PVLDZFRELLNELLEALKQKLK32%  58 67 68 62  35 (SEQ ID NO: 35) PVLDWFRELLNELLEALKQKLK 31%  49 (sp)59 61  36 (SEQ ID NO: 36) PLLELLKELLQELLEALKQKLK 31%  95 100 95  37 (SEQID NO: 37) PVLDLFREWLNELLEALKQKLK 29%  65 75 76 73  38 (SEQ ID NO: 38)PVLDLFRELLNEXLEAWKQKLK 29%  25 49 21 49  39 (SEQ ID NO: 39)PVLDLFRELLEELLKALKKKLK 25%  66 69 68 72  40 (SEQ ID NO: 40)PVLDLFNELLRELLEALQKKLK 25%  66 84 79 77  41 (SEQ ID NO: 41)PVLDLWRELLNEXLEALKQKLK 25%  53 73 85 69  42 (SEQ ID NO: 42)PVLDEFREKLNEXWEALKQKLK 25%  15 74 27 76  43 (SEQ ID NO: 43)PVLDEFREKLWEXLEALKQKLK 25%  44 (SEQ ID NO: 44) pvldefreklfleXlealkqklk25%  20 86  45 (SEQ ID NO: 45) PVLDEFREKLNEXLEALKQKLK 24%  24 84 25 86 46 (SEQ ID NO: 46) PVLDLFREKLNEXLEALKQKLK 23%  30 86 58 85  47 (SEQ IDNO: 47) ˜VLDLFRELLNEGLEALKQKLK 23%  48 (SEQ ID NO: 48)pVLDLFRELLNELLEALKQKLK 22%  49 (SEQ ID NO: 49) PVLDLFRNLLEKLLEALEQKLK22%  57 65 52 57  50 (SEQ ID NO: 50) PVLDLFRELLWEXLEALKQKLK 21%  68 8489 76  51 (SEQ ID NO: 51) PVLDLFWELLNEXLEALKQKLK 20%  63 82 81 73  52(SEQ ID NO: 52) PVWDEFREKLNEXLEALKQKLK 20% sp sp sp  53 (SEQ ID NO: 53)VVLDLFRELLNELLEALKQKLK 19%  54 (SEQ ID NO: 54) PVLDLFRELLNEWLEALKQKLK19%  76 71 84 78  55 (SEQ ID NO: 55) P˜˜˜LFRELLNELLEALKQKLK 19%  38 7278 75  56 (SEQ ID NO: 56) PVLDLFRELLNELLEALKQKKK 18%  57 (SEQ ID NO: 57)PVLDLFRNLLEELLKALEQKLK 18%  58 (SEQ ID NO: 58) PVLDEFREKLISTEXLEALKQKL18%  59 (SEQ ID NO: 59) LVLDLFRELLNELLEALKQKLK 17%  60 (SEQ ID NO: 60)PVLDLFRELLNELLEALKQ˜˜˜ 16%  39 83 66 84  61 (SEQ ID NO: 61)PVLDEFRWKLNEXLEALKQKLK 16%  62 (SEQ ID NO: 62) PVLDEWREKLNEXLEALKQKLK16%  15 85 43  63 (SEQ ID NO: 63) PVLDFFREKLNEXLEALKQKLK 16%  64 (SEQ IDNO: 64) PWLDEFREKLNEXLEALKQKLK 15%  65 (SEQ ID NO: 65)˜VLDEFREKLNEXLEALKQKLK 15%  66 (SEQ ID NO: 66) PVLDLFRNLLEELLEALQKKLK15%  64 82 66 70  67 (SEQ ID NO: 67) ˜VLDLFRELLNELLEALKQKLK 14%  81 9084 94  68 (SEQ ID NO: 68) PVLDEFRELLKEXLEALKQKLK 14%  69 (SEQ ID NO: 69)PVLDEFRKKLNEXLEALKQKLK 13%  70 (SEQ ID NO: 70) PVLDEFRELLYEXLEALKQKLK12%  27 78 33 66  71 (SEQ ID NO: 71) PVLDEFREKLNELXEALKQKLK 11%  72 (SEQID NO: 72) PVLDLFRELLNEXLWALKQKLK 11% sp sp sp  73 (SEQ ID NO: 73)PVLDEFWEKLNEXLEALKQKLK 10%  74 (SEQ ID NO: 74) PVLDKFREKLNEXLEALKQKLK10%  75¹ (SEQ ID NO: 75) PVLDEFREKLNEELEALKQKLK 10%  18 28 23 55  76(SEQ ID NO: 76) PVLDEFRELLFEXLEALKQKLK 9%  41 88 66  77 (SEQ ID NO: 77)PVLDEFREKLNKXLEALKQKLK 9%  78 (SEQ ID NO: 78) PVLDEFRDKLNEXLEALKQKLK  79(SEQ ID NO: 79) PVLDEFRELLNELLEALKQKLK 9%  80 (SEQ ID NO: 80)PVLDLFERLLNELLEALQKKLK 9%  81 (SEQ ID NO: 81) PVLDEFREKLNWXLEALKQKLK  82(SEQ ID NO: 82) ˜˜LDEFREKLNEXLEALKQKLK 8%  83 (SEQ ID NO: 83)PVLDEFREKLNEXLEALWQKLK  84 (SEQ ID NO: 84) PVLDEFREKLNELLEALKQKLK 7%  85(SEQ ID NO: 85) P˜LDLFRELLNELLEALKQKLK 7%  58 61 64 69  86 (SEQ ID NO:86) PVLELFERLLDELLNALQKKLK 7%  87 (SEQ ID NO: 87) pllellkellqellealkqklk7% 100 100 100  88 (SEQ ID NO: 88) PVLDKFRELLNEXLEALKQKLK 7%  89 (SEQ IDNO: 89) PVLDEFREKLNEXLWALKQKLK 6%  90 (SEQ ID NO: 90)˜˜˜DEFREKLNEXLEALKQKLK 6%  91 (SEQ ID NO: 91) PVLDEFRELLNEXLEALKQKLK 6% 43 100 100  92 (SEQ ID NO: 92) PVLDEFRELYNEXLEALKQKLK 5%  93 (SEQ IDNO: 93) PVLDEFREKLNEXLKALKQKLK 5%  94² (SEQ ID NO: 94)PVLDEFREKLNEALEALKQKLK 5%  18 70 27 63  95 (SEQ ID NO: 95)PVLDLFRELLNLXLEALKQKLK 5% sp sp  96 (SEQ ID NO: 96)pvldlfrellneXlealkqklk 5%  52 85 63 81  97 (SEQ ID NO: 97)PVLDLFRELLNELLE˜˜˜˜˜˜˜ 4%  98 (SEQ ID NO: 98) PVLDLFRELLNEELEALKQKLK 2% 99 (SEQ ID NO: 99) KLKQKLAELLENLLERFLDLVP 2%  72 88 80 80 100 (SEQ IDNO: 100) pvldlfrellnellealkqklk 2%.  83 92 98 101 (SEQ ID NO: 101)PVLDLFRELLNWXLEALKQKLK 2% sp sp 102 (SEQ ID NO: 102)PVLDLFRELLNLXLEALKEKLK 2% sp 103 (SEQ ID NO: 103) PVLDEFRELLNEELEALKQKLK1% 104 (SEQ ID NO: 104) P˜˜˜˜˜˜˜LLNELLEALKQKLK 1%  21 49 29 55 105 (SEQID NO: 105) PAADAFREAANEAAEAAKQKAK 1%  29 28 32 65 106 (SEQ ID NO: 106)PVLDLFREKLNEELEALKQKLK 0% 107 (SEQ ID NO: 107) klkqklaellenllerfldlvp 0%sp sp 77 108 (SEQ ID NO: 108) PVLDLFRWLLNEXLEALKQKLK 0%  28 55 54 109³(SEQ ID NO: 109) PVLDEFREKLNERLEALKQKLK 0%  19 45 23 58 110 (SEQ ID NO:110) PVLDEFREKLNEXXEALKQKLK 0% 111 (SEQ ID NO: 111)PVLDEFREKLWEXWEALKQKLK 0% 112 (SEQ ID NO: 112) PVLDEFREKLNEXSEALKQKLK 0%113 (SEQ ID NO: 113) PVLDEFREKLNEPLEALKQKLK 0%  6 22 114 (SEQ ID NO:114) PVLDEFREKLNEXMEALKQKLK 0% 115 (SEQ ID NO: 115PKLDEFREKLNEXLEALKQKLK 0% 116 (SEQ ID NO: 116) PHLDEFREKLNEXLEALKQKLK 0%117 (SEQ ID NO: 117) PELDEFREKLNEXLEALKQKLK 0% 118 (SEQ ID NO: 118)PVLDEFREKLNEXLEALEQKLK 0% 119 (SEQ ID NO: 119) PVLDEFREKLNEELEAXKQKLK 0%120 (SEQ ID NO: 120) PVLDEFREKLNEELEXLKQKLK 0% 121 (SEQ ID NO: 121)PVLDEFREKLNEELEALWQKLK 0% 122 (SEQ ID NO: 122) PVLDEFREKLNEELEWLKQKLK 0%123 (SEQ ID NO: 123) QVLDLFRELLNELLEALKQKLK 124 (SEQ ID NO: 124)PVLDLFOELLNELLEALOQOLO 125 (SEQ ID NO: 125) NVLDLFRELLNELLEALKQKLK 126(SEQ ID NO: 126) PVLDLFRELLNELGEALKQKLK 127 (SEQ ID NO: 127)PVLDLFRELLNELLELLKQKLK 47% 128 (SEQ ID NO: 128) PVLDLFRELLNELLEFLKQKLK129 (SEQ ID NO: 129) PVLELFNDLLRELLEALQKKLK 130 (SEQ ID NO: 130)PVLELFNDLLRELLEALKQKLK 131 (SEQ ID NO: 131) PVLELFKELLNELLDALRQKLK 132(SEQ ID NO: 132) PVLDLFRELLENLLEALQKKLK 133 (SEQ ID NO: 133)PVLELFERLLEDLLQALNKKLK 134 (SEQ ID NO: 134) PVLELFERLLEDLLKALNQKLK 135(SEQ ID NO: 135) DVLDLFRELLNELLEALKQKLK 136 (SEQ ID NO: 136)PALELFKDLLQELLEALKQKLK 137 (SEQ ID NO: 137) PVLDLFRELLNEGLEAZKQKLK 138(SEQ ID NO: 138) PVLDLFRELLNEGLEWLKQKLK 139 (SEQ ID NO: 139)PVLDLFRELWNEGLEALKQKLK 140 (SEQ ID NO: 140) PVLDLFRELLNEGLEALOQOLO 141(SEQ ID NO: 141) PVLDFFRELLNEGLEALKQKLK 142 (SEQ ID NO: 142)PVLELFRELLNEGLEALKQKLK 143 (SEQ ID NO: 143) PVLDLFRELLNEGLEALKQKLK* 144(SEQ ID NO: 144) pVLELFEWLLERLLDALQKKLK 111%  89 88 95 145 (SEQ ID NO:145) GVLELFENLLERLLDALQKKLK 100%  55 51 58 146 (SEQ ID NO: 146)PVLELFENLLERLLDALQKKLK 86%  97 100 100 95 147 (SEQ ID NO: 147)PVLELFENLLERLFDALQKKLK 76% 148 (SEQ ID NO: 148) PVLELFENLLERLGDALQKKLK75%  10 76 23 80 149 (SEQ ID NO: 149) PVLELFENLWERLLDALQKKLK 63%  28 5447 150 (SEQ ID NO: 150) PLLELFENLLERLLDALQKKLK 57% 151 (SEQ ID NO: 151)PVLELFENLGERLLDALQKKLK 55% 152 (SEQ ID NO: 152) PVFELFENLLERLLDALQKKLK50% 153 (SEQ ID NO: 153) AVLELFENLLERLLDALQKKLK 49% 154 (SEQ ID NO: 154)PVLELFENLLERGLDALQKKLK 39%  13 76 25 80 155 (SEQ ID NO: 155)PVLELFLNLWERLLDALQKKLK 38% 156 (SEQ ID NO: 156) PVLELFLNLLERLLDALQKKLK35% 157 (SEQ ID NO: 157) PVLEFFENLLERLLDALQKKLK 30% 158 (SEQ ID NO: 158)PVLELFLNLLERLLDWLQKKLK 30% 159 (SEQ ID NO: 159) PVLDLFENLLERLLDALQKKLK28% 160 (SEQ ID NO: 160) PVLELFENLLERLLDWLQKKLK 28%  65 73 75 61 161(SEQ ID NO: 161) PVLELFENLLERLLEALQKKLK 27% 162 (SEQ ID NO: 162)PVLELFENWLERLLDALQKKLK 27%  68 83 81 163 (SEQ ID NO: 163)PVLELFENLLERLWDALQKKLK 26%  27 53 55 164 (SEQ ID NO: 164)PVLELFENLLERLLDAWQKKLK 24%  37 66 51 61 165 (SEQ ID NO: 165)PVLELFENLLERLLDLLQKKLK 23% 166 (SEQ ID NO: 166) PVLELFLNLLEKLLDALQKKLK22% 167 (SEQ ID NO: 167) PVLELFENGLERLLDALQKKLK 18% 168 (SEQ ID NO: 168)PVLELFEQLLEKLLDALQKKLK 17% 169 (SEQ ID NO: 169) PVLELFENLLEKLLDALQKKLK17% 170 (SEQ ID NO: 170) PVLELFENLLEOLLDALQOOLO 17% 171 (SEQ ID NO: 171)PVLELFENLLEKLLDLLQKKLK 16% 172 (SEQ ID NO: 172) PVLELFLNLLERLGDALQKKLK16% 173 (SEQ ID NO: 173) PVLDLFDNLLDRLLDLLNKKLK 15% 174 (SEQ ID NO: 174)pvlelfenllerlldalqkklk 13% 175 (SEQ ID NO: 175) PVLELFENLLERLLELLNKKLK13% 176 (SEQ ID NO: 176) PVLELWENLLERLLDALQKKLK 11% 177 (SEQ ID NO: 177)GVLELFLNLLERLLDALQKKLK 10% 178 (SEQ ID NO: 178) PVLELFDNLLEKLLEALQKKLR9% 179 (SEQ ID NO: 179) PVLELFDNLLERLLDALQKKLK 8% 180 (SEQ ID NO: 180)PVLELFDNLLDKLLDALQKKLR 8% 181 (SEQ ID NO: 181) PVLELFENLLERWLDALQKKLK 8%182 (SEQ ID NO: 182) PVLELFENLLEKLLEALQKKLK 7% 183 (SEQ ID NO: 183)PLLELFENLLEKLLDALQKKLK 6% 184 (SEQ ID NO: 184) PVLELFLNLLERLLDAWQKKLK 4%185 (SEQ ID NO: 185) PVLELFENLLERLLDALQOOLO 3% 186 (SEQ ID NO: 186)PVLELFEQLLERLLDALQKKLK 187 (SEQ ID NO: 187) PVLELFENLLERLLDALNKKLK 188(SEQ ID NO: 188) PVLELFENLLDRLLDALQKKLK 189 (SEQ ID NO: 189)DVLELFENLLERLLDALQKKLK 190 (SEQ ID NO: 190) PVLEFWDNLLDKLLDALQKKLR 191(SEQ ID NO: 191) PVLDLLRELLEELKQKLK* 100% 192 (SEQ ID NO: 192)PVLDLFKELLEELKQKLK* 100%  36 56 193 (SEQ ID NO: 193) PVLDLFRELLEELKQKLK*96%  34 88 87 87 194 (SEQ ID NO: 194) PVLELFRELLEELKQKLK* 88%  38 93 93195 (SEQ ID NO: 195) PVLELFKELLEELKQKLK* 87% 196 (SEQ ID NO: 196)PVLDLFRELLEELKNKLK* 81% 197 (SEQ ID NO: 197) PLLDLFRELLEELKQKLK* 81%  4370 69 198 (SEQ ID NO: 198) GVLDLFRELLEELKQKLK* 80% 199 (SEQ ID NO: 199)PVLDLFRELWEELKQKLK* 76%  35 77 80 79 200 (SEQ ID NO: 200)NVLDLFRELLEELKQKLK* 75% 201 (SEQ ID NO: 201) PLLDLFKELLEELKQKLK* 74% 202(SEQ ID NO: 202) PALELFKDLLEELRQKLR* 70% 203 (SEQ ID NO: 203)AVLDLFRELLEELKQKLK* 66% 204 (SEQ ID NO: 204) PVLDFFRELLEELKQKLK* 63% 205(SEQ ID NO: 205) PVLDLFREWLEELKQKLK* 60% 206 (SEQ ID NO: 206)PLLELLKELLEELKQKLK* 57% 207 (SEQ ID NO: 207) PVLELLKELLEELKQKLK* 50% 208(SEQ ID NO: 208) PALELFKDLLEELRQRLK* 48% 209 (SEQ ID NO: 209)PVLDLFRELLNELLQKLK 47%  54 71 67 62 210 (SEQ ID NO: 210)PVLDLFRELLEELKQKLK 46%  20 63 37 53 211 (SEQ ID NO: 211)PVLDLFRELLEELOQOLO* 45% 212 (SEQ ID NO: 212) PVLDLFOELLEELOQOLK* 43% 213(SEQ ID NO: 213) PALELPKDLLEEFRQRLK* 42% 214 (SEQ ID NO: 214)pVLDLFRELLEELKQKLK* 39% 215 (SEQ ID NO: 215) PVLDLFRELLEEWKQKLK* 38%  2863 53 68 216 (SEQ ID NO: 216) PVLELFKELLEELKQKLK 35% 217 (SEQ ID NO:217) PVLDLFRELLELLKQKLK 30%  52 78 76 70 218 (SEQ ID NO: 218)PVLDLFRELLNELLQKLK* 29% 219 (SEQ ID NO: 219) PVLDLFRELLNELWQKLK 24% 220(SEQ ID NO: 220) PVLDLFRELLEELQKKLK 22%  27 64 54 64 221 (SEQ ID NO:221) DVLDLFRELLEELKQKLK* 12% 222 (SEQ ID NO: 222) PVLDAFRELLEALLQLKK 8%223 (SEQ ID NO: 223) PVLDAFRELLEALAQLKK 8%  21 56 23 51 224 (SEQ ID NO:224) PVLDLFREGWEELKQKLK 8% 225 (SEQ ID NO: 225) PVLDAFRELAEALAQLKK 1%226 (SEQ ID NO: 226) PVLDAFRELGEALLQLKK 1% 227 (SEQ ID NO: 227)PVLDLFRELGEELKQKLK* 0% 228 (SEQ ID NO: 228) PVLDLFREGLEELKQKLK* 0% 229(SEQ ID NO: 229) PVLDLFRELLEEGKQKLK* 0% 230 (SEQ ID NO: 230)PVLELFERLLEDLQKKLK 231 (SEQ ID NO: 231) PVLDLFRELLEKLEQKLK 232 (SEQ IDNO: 232) PLLELFKELLEELKQKLK* 237⁴ (SEQ ID NO: 237) LDDLLQKWAEAFNQLLKK11%  30 66 45 — 238⁵ (SEQ ID NO: 238) EWLKAFYEKVLEKLKELF* 19%  49 72 6058 239⁶ (SEQ ID NO: 239) EWLEAFYKKVLEKLKELF* 11%  44 49 sp 240 (SEQ IDNO: 240) DWLKAFYDKVAEKLKEAF* 10%  16 68 59 57 241 (SEQ ID NO: 241)DWFKAFYDKVFEKFKEFF 8% 242⁷ (SEQ ID NO: 242) GIKKFLGSIWKFIKAFVG 7% 243(SEQ ID NO: 243) DWFKAFYDKVAEKFKEAF 5%  10 64 50 244⁸ (SEQ ID NO: 244)DWLKAFYDKVAEKLKEAF 5%  9 40 13 48 245 (SEQ ID NO: 245)DWLKAPYDKVFEKFKEFF 4%  38 77 70 sp 246⁹ (SEQ ID NO: 246)EWLEAFYKKVLEKLKELF 4%  18 44 47 247 (SEQ ID NO: 247) DWFKAFYDKFFEKFKEFF3% 248¹⁰ (SEQ ID NO: 248) EWLKAFYEKVLEKLKELF 3%  18 45 13 249¹¹ (SEQ IDNO: 249) EWLKAEYEKVEEKLKELF* 250¹² (SEQ ID NO: 250) EWLKAEYEKVLEKLKELF*251¹³ (SEQ ID NO: 251) EWLKAFYKKVLEKLKELF* 252 (SEQ ID NO: 252)PVLDLFRELLEQKLK* 253 (SEQ ID NO: 253) PVLDLFRELLEELKQK* 254 (SEQ ID NO:254) PVLDLFRELLEKLKQK* 255 (SEQ ID NO: 255) PVLDLFRELLEKLQK* 256 (SEQ IDNO: 256) PVLDLFRELLEALKQK* 257 (SEQ ID NO: 257) PVLDLFENLLERLKQK* 258(SEQ ID NO: 258) PVLDLFRELLNELKQK*¹Segrest's Consensus 22-mer peptide (Anantharamaiah et al., 1990,Arteriosclerosis 10(1): 95-105).²[A¹³]-Consensus 22-mer peptide (Anantharamaiah et al., 1990,Arteriosclerosis 10(1): 95-105).³[R¹³]-Consensus 22-mer peptide (Anantharamaiah et al., 1990,Arteriosclerosis 10(1): 95-105).⁴ID-3 peptide (Labeur et al., 1997, Arteriosclerosis, Thrombosis andVascular Biology 17(3): 580-588).⁵Ac-18AMOD-C(O)NH₂ peptide (Epand et al., 1987, J. Biol. Chem. 262(19):9389-9396).⁶Ac-18AM4-C(O)NH₂ peptide (Brasseur, 1993, Biochim. Biophys. Acta 1170:1-7).⁷18L peptide (Segrest et al., 1990, Proteins: Structure, Function andGenetics 8: 103-117).⁸18A peptide (Anantharamaiah et al., 1985, J. Biol. Chem. 260(18):10248-10255).⁹18AM4 peptide (Rosseneu et al., W093/25581; Corijn et al., 1993,Biochim. Biophys. Acta 1170: 8-16).¹⁰[Glu^(1,8); Leu^(5,11,17)] 18A peptide (Epand et al., 1987, J. Biol.Chem. 262(19): 9389-9396).¹¹Ac-18AM3-C(O)NH₂ (Rosseneu et al., W093/25581).¹²Ac-18AM2-C(O)NH₂ (Rosseneu et al., W093/25581).¹³Ac-18AM1-C(O)NH₂ (Rosseneu et al., W093/25581).

In TABLE X, * indicates peptides that are N-terminal acetylated andC-terminal amidated; ′ indicates peptides that are N-terminaldansylated; sp indicates peptides that exhibited solubility problemsunder the experimental conditions; X is Aib; Z is Nal; O is Orn; He (%)designates percent helicity; mics designates micelles; and ˜ indicatesdeleted amino acids.

7. EXAMPLE: STRUCTURAL AND LIPID BINDING ANALYSIS OF APoA-I PEPTIDES

The structural and lipid binding characteristics of the purifiedpeptides synthesized as described in Section 6, supra, were determinedby circular dichroism (CD), fluorescence spectroscopy and nuclearmagnetic resonance (NMR).

7.1. CIRCULAR DICHROISM

This Example describes a preferred method for determining the percentageof α-helical secondary structure of the peptides alone and in thepresence of lipids.

7.1.1 EXPERIMENTAL METHOD

Far UV circular dichroism spectra were recorded between 190 and 260 nm(in 0.5 nm or 0.2 nm increments) with a AVIV62DS spectrometer (AVIVAssociates, Lakewood, N.J., USA) equipped with a thermoelectric cellholder and sample changer. The instrument was calibrated with(+)-10-camphoric acid. Between one and three scans were collected foreach sample, using 10 cm, 5 cm, 1 cm and 0.1 cm path length quartzSuprasil cells, respectively, for peptide concentrations of 10⁻⁷ M to10⁻⁴ M. The bandwidth was fixed at 1.5 nm and the scan speed to 1s perwavelength step. The reported data are the mean of at least 2 or 3independent measurements.

After background substraction, spectra were converted to molarellipticity (e) per residue in deg. cm⁻² dmol⁻¹. The peptideconcentration was determined by amino acid analysis and also byabsorption spectrometry on a Perkin Elmer Lambda 17 UV/Visiblespectrophotometer when the peptide contained a chromophore (tryptophane,dansyl, naphtylalanine).

CD spectra were obtained with free, unbound peptide (5 μM in 5 mMphosphate buffer, pH 7.4); with peptide-SUV complexes (20:1 EPC:Chol.,Ri=50); with peptide-micelle complexes(1-myristoyl-2-hydroxy-sn-glycero-3-phosphatidyl choline, Ri=100); andwith free, unbound peptide in the presence of 2,2,2-trifluoroethanol(TFE) (5 μM peptide, 90% vol TFE).

The SUVs were obtained by dispersing the lipids (10 mM, 20:1 EPC:Chol.,Avanti Polar Lipids, AL) in phosphate buffer (5 mM, pH 7.4) withbubbling N₂ for 5 min., followed by sonication (1.5 hr.) in a bathsonicator. The homogeneity of the preparation was checked by FPLC.

The micelles were obtained by dispersing the lipid (6 mM1-myristoyl-2-hydroxy-sn-glycero-3-phosphatidyl choline, Avanti PolarLipids, AL) in phosphate buffer (5 mM, pH 7.4) with bubbling N₂ for 5min., followed by vortexing.

To obtain the peptide-SUV complexes, SUVs were added to the peptide (5μM in 5 mM phosphate buffer, pH 7.4) at a phospholipid-peptide molarratio (Ri) of 100.

To obtain the peptide-micelle complexes, micelles were added to thepeptide (5 μM in 5 mM phosphate buffer, pH 7.4) at a Ri of 100.

All spectra were recorded at 37° C. The stability of peptide 210 (SEQ IDNO:210) as a function of temperature (both free in buffer and inmicelles) was determined by recording spectra at a series of differenttemperatures.

The degree of helicity of peptide 210 (SEQ ID NO:210) as a function ofconcentration was also determined.

7.1.2 HELICITY DETERMINATION

The degree of helicity of the peptides in the various conditions wasdetermined from the mean residue ellipticity at 222 nm (Chen et al.,1974, Biochemistry 13:3350-3359) or by comparing the CD spectra obtainedto reference spectra available on databases (16 helical referencespectra from Provencher & Glockner, 1981, Biochemistry 20:33-37;denatured protein reference spectra from Venyaminov et al., 1993, Anal.Biochem. 214:17-24) using the CONTIN curve-fitting algorithm version2DP, CD-1 pack (Aug. 1982) (Provencher, 1982, Comput. Phys. Commun.27:213-227, 229-242). Acceptable fit was determined using thestatistical analysis methodology provided by the CONTIN algorithm. Theerror of all methods was ±5% helicity.

7.1.3 RESULTS

The degree of helicity (%) of the free, unbound peptides (free), thepeptide-SUV complexes (SUVs), the peptide-micelle complexes (mics) andthe peptide-TFE solution (TFE) are reported in TABLE X, Section 8.3,infra.

Peptide 210 (SEQ ID NO:210) contains significant α-helical structure(63% helicity) in micelles. Moreover, the α-helical structure iscompletely stable over a temperature range of 5°-45° C. (data notshown). The helicity of peptide 210 (SEQ ID NO:210) also increases inthe presence of TFE, which is a solvent that, due to having asignficantly lower dielectric constant (ε=26.7) than water (ε=78.4),stabilizes α-helices and intrapeptide hydrogen bonds at concentrationsbetween 5-90% (v/v).

Referring to TABLE VIII, infra, it can be seen that those peptides whichexhibit a high degree of LCAT activation (≧38%) generally possesssignificant α-helical structure in the presence of lipids (≧60% helicalstructure in the case of unblocked peptides containing 22 or more aminoacids or blocked peptides containing 18 or fewer amino acids; ≧40%helical structure in the case of unblocked peptides containing 18 orfewer amino acids), whereas peptides which exhibit little or no LCATactivation possess little α-helical structure. However, in someinstances, peptides which contain significant α-helical structure in thepresence of lipids do not exhibit significant LCAT activation. As aconsequence, the ability of the core peptides of the invention to adoptan α-helical structure in the presence of lipids is considered acritical feature of the core peptides of the invention, as the abilityto form an α-helix in the presence of lipids. appears to be aprerequisite for LCAT activation.

7.2 FLUORESCENCE SPECTROSCOPY

The lipid binding properties of the peptides described in Section 6,supra, were tested by fluorescence measurements with labeled peptides,in the present case Tryptophane (Trp or W) or Naphtylalanine (Nal). Thefluorescence spectra were recorded on a Fluoromax from Spex (Jobin-Yvon)equipped with a Xenon lamp of 150W, two monochromators (excitation andemission), a photomultiplier R-928 for detection sensitive in the red upto 850 nm and a thermoelectric magnetic stirred cell holder. QuartzSuprasil cuvettes were used for measurements in the micromolarconcentration range. A device of variable slits (from 0.4 to 5 nm)allows modulation of the incident and emitted intensities according tothe concentration of peptide used. The reported values are in generalthe average of between 2 to 4 spectra. The peptide concentration isdetermined by absorption spectrometry on a Philips PU 8800 using theabsorption band of the Trp (ε_(280 nm)=5,550 M⁻¹cm⁻¹ in Tris buffer) orthe Nal (ε_(224 nm)=92, 770 M⁻¹ cm⁻³ in methanol).

Fluorescence spectra of the peptides were recorded between 290 nm and450 nm in Tris-HCl buffer (20 mM, pH=7.5), in the presence and absenceof lipidic vesicles. The small unilamellar vesicles were formed afterrehydration in buffer of the lyophilized phospholipids, dispersion andtip sonification under a N₂ stream. The lipids used were either EggPC/Chol. (20:1) or POPC/Chol. (20:1). The spectra were recorded at apeptide concentration of 2 μM and at a temperature of 37° C. Thefluorescence reference standard in the case of Trp wasN-acetyltryptophanylamide (NATA).

Lipid binding studies were done through progressive lipidic vesicleaddition to the peptide in solution at 2 μM (slits:5 nm in excitationand 1.5 nm in emission). Dilution effects were taken into account forthe fluorescence intensity determination. The lipid concentrations werevaried from 10 to 600 μM and the molar ratio of lipid to peptide (Ri)was varied from 5 to 300. The wavelength of excitation was set at 280 nmfor both Trp and Nal.

7.2.1 FLUORESCENCE SPECTRAL ANALYSIS

The data were directly recorded and treated by an IBM-PC linked to thespectrofluorimeter through the DM3000F software from Spex. The spectrawere corrected by substraction of the solvent contribution and byapplication of a coefficient given by the constructor taking intoaccount the variation of the photomultiplier response versus thewavelength.

The fluorescence spectra of the peptides were characterized by thewavelength at their maximum of fluorescence emission and by theirquantum yield compared to NATA in the case of peptides labeled with atryptophane. The process of binding to lipids was analyzed bycalculating the shift of the wavelength at the maximum of fluorescenceemission, (λ_(max)), and the variation of the relative fluorescenceintensity of emission versus the lipid concentration. The relativefluorescence intensity is defined as the following ratio:(I−I₀)_(λmax)/I_(0λmax). I and I₀ are both measured at the (λ_(max))corresponding to the initial free state of the peptide, i.e., withoutlipids. I is the intensity at a defined lipid to peptide ratio and I₀ isthe same parameter measured in absence of lipids. The absence of thesevariations is relevant of the absence of interactions of the peptideswith the lipids.

7.2.2 RESULTS AND DISCUSSION

The lipid binding properties of peptide 199 (SEQ ID NO:199), which issimilar in primary sequence to peptide 210 (SEQ ID NO:210) except thatit contains a W (Trp) residue at position 10, are presented in TABLE IX.TABLE IX BINDING PROPERTIES OF PEPTIDE 199 (SEQ ID NO: 199) TO LIPIDICVESICLES AS MEASURED BY FLUORESCENCE Lipid:Peptide Molar Ratio (Ri)I/I_(o) λ_(max) (nm) 0 0 348 5 8 344 10 8 339 30 18 328 60 22 100 27 326200 41 325

In buffer at a concentration of 2 μm, the maximum of the tryptophanefluorescence emission (λ_(max)) of peptide 199 (SEQ ID NO:199) is 348nm. This corresponds to a tryptophane which is relatively exposed to theaqueous environment when compared to NATA (λ max=350 nm). Peptide 199(SEQ ID NO:199) binds very effectively to EPC/Chol (20:1) smallunilamellar vesicles as demonstrated by the burying of the tryptophane(the wavelength for the tryptophane maximum fluorescence emission shiftsfrom 348 nm to 325 nm) and the high fluorescence intensity exaltation(see Table IX). The burying of the tryptophane residue is maximal for alipid to peptide molar ratio of about 100.

Other peptides which exhibited a high degree of helicity in the presenceof lipids (≧60% for unblocked peptides of ≧22 acids, or blocked peptidesof ≦18 amino acids; ≧40% for unblocked peptides of ≦18 amino acids) asmeasured by circular dichroism as disclosed in Section 7.1, supra, alsodemonstrated good lipid binding. Of course, among all the peptidesselected by the circular dichroism screening, only the ones that couldbe followed by fluorescence were tested for their lipid bindingproperties.

7.3 NUCLEAR MAGNETIC RESONANCE (NMR)

This Example describes an NMR method for analyzing the structure of thecore peptides of the invention.

7.3.1 NMR SAMPLE PREPARATION

Samples were prepared by dissolving 5 mg of peptide in 90% H₂O/10% D₂Ocontaining trace amounts of 2,2-Dimethyl-2-sila-5-pentane sulfonate(DSS) as an internal chemical shift reference. Some of the samplescontained trifluoroethanol (TFE) (expressed as % vol). The total samplevolume was 500 μl and the concentration of peptide was approximately 5mM.

7.3.2 NMR SPECTROSCOPY

¹H NMR spectra were acquired at 500 MHz using a Bruker DRX500spectrometer equipped with a B-VT2000 temperature control unit. One andtwo-dimensional experiments were recorded using standard pulsesequences. (Two Dimensional NMR Spectroscopy, Eds. W. R. Croasmun andRMK Carlson, 1994, VCH Publishers, New York, USA). Water suppression wasachieved with low power presaturation for 2 sec. Two-dimensionalexperiments were carried out in the phase sensitive mode using timeproportional phase incrementation (TPPI) and a spectral width of 6000 Hzin both dimensions. Typically, 40 scans were co-added for 400 t₁increments with 2048 data points. Data were processed using FELIX95software (Molecular Simulations) on an INDIGO2 workstation (SiliconGraphics). Data were zero-filled to give a 2K×2K data matrix andapodized by a 450 shifted squared sine-bell function.

7.3.3 NMR ASSIGNMENT

Complete proton resonance assignments were obtained by applying thesequential assignment technique using DQFCOSY, TOCSY and NOESY spectraas described in the literature (Wuthrich, NMR of Proteins and NucleicAcids, 1986, John Wiley & Sons, New York, USA). Secondary chemicalshifts were calculated for HN and Hα protons by subtracting thetabulated random coil chemical shifts (Wishart and Sykes, 1994, Method.Enz. 239:363-392) from the corresponding experimental values.

7.3.4 RESULTS AND DISCUSSION

General Consideration. Amphipathic helical peptides tend to aggregate inaqueous solutions at the high concentrations necessary for NMRspectroscopy, making it difficult to obtain high resolution spectra. TFEis known to solubilize peptides, and in addition stabilizes helicalconformations of peptides having helical propensity. The findings fromNMR spectroscopy are demonstrated for peptide 210 (SEQ ID NO:210) as arepresentative example. The consensus 22-mer of Segrest (SEQ ID NO:75)was studied in comparison.

Secondary chemical shifts. Proton chemical shifts of amino acids dependboth on the type of residue and on the local secondary structure withina peptide or protein (Szlagyi, 1995, Progress in Nuclear MagneticResonance Spectroscopy 27:325-443). Therefore, identification of regularsecondary structure is possible by comparing experimental shifts withtabulated values for random coil conformation.

Formation of an α-helix typically results in an up-field (negative)shift for the Hα resonance. Observation of an upfield Hα shift forseveral sequential residues is generally taken as evidence of a helicalstructure. The Hα secondary shifts for peptide 210 (SEQ ID NO:210) in25% TFE at 295 K show a significant negative shift for residues 4through 15, demonstrating a highly helical conformation. Smalldifferences are observed in the Hα chemical shifts of the consensus22-mer (SEQ ID NO:75) compared to peptide 210 (SEQ ID NO:210).

The chemical shifts of amide hydrogens of amino acid residues residingin regions of α-helix are also shifted upfield with respect to thechemical shifts observed for random coil. In addition, a periodicity ofthe HN shifts can be observed, and it reflects the period of the helicalturns. The amplitude of the shift variation along the sequence isrelated to the amphipathicity of a helical peptide. A higher hydrophobicmoment leads to a more pronounced oscillation (Zhou et al., 1992, J. Am.Chem. Soc. 114:4320-4326). The HN secondary shifts for peptide 210 (SEQID NO:210) in 25% TFE at 295 K show an oscillatory behavior in agreementwith the amphipathic nature of the helix.

The amino acid replacements lead to a more pronounced periodicity alongthe entire sequence. The pattern clearly reflects the strongeramphipathic nature of peptide 210 (SEQ ID NO:210) as compared toSegrest's consensus 22-mer (SEQ ID NO:75). The existence of 4-5 helicalturns can be discerned.

The secondary shift of an amide proton is influenced by the length ofthe hydrogen bond to the carbonyl oxygen one turn away from the helix.Therefore, the periodicity of observed chemical shift values reflectsdifferent hydrogen bond lengths. This difference is associated with anoverall curved helical shape of the helix backbone. The hydrophobicresidues are situated on the concave side. The secondary shifts ofpeptide 210 (SEQ ID NO:210) indicate a curved α-helical conformation.

8 EXAMPLE: PHARMACOKINETICS OF THE ApoA-I AGONISTS

The following experiments can be used to demonstrate that the ApoA-Iagonists are stable in the circulation and associate with the HDLcomponent of plasma.

8.1 SYNTHESIS OF RADIOLABELLED PEPTIDES

Radiolabelled peptides are synthesized by coupling a ¹⁴C-labeled aminoacid as the N-terminal amino acid. The synthesis is carried outaccording to Lapatsanis, Synthesis, 1983, 671-173. Briefly, 250 μM ofunlabeled N-terminal amino acid is dissolved in 225 μl of a 9% Na₂CO₃solution and added to a solution (9% Na₂CO₃) of 9.25 MBq (250 ∥M)¹⁴C-labeled N-terminal amino acid. The liquid is cooled down to 0° C.,mixed with 600 μM (202 mg) 9-fluorenylmethyl-N-succihimidylcarbonate(Fmoc-OSu) in 0.75 ml DMF and shaken at room temperature for 4 hr.Thereafter the mixture is extracted with Diethylether (2×5 ml) andchloroform (1×5 ml), the remaining aqueous phase is acidified with 30%HCl and extracted with chloroform (5×8 ml). The organic phase is driedover Na₂SO₄, filtered off and the volume was reduced under nitrogen flowto 5 ml. The purity is estimated by TLC (CHCl₃:MeOH:Hac, 9:1:0.1 v/v/v,stationary phase RPTLC silicagel 60, Merck, Germany).

8.2 PHARMACOKINETICS IN MICE

In each experiment, 2.5 mg/kg radiolabelled peptide is injectedintraperitoneally into mice which are fed normal mouse chow or theatherogenic Thomas-Harcroft modified diet (resulting in severelyelevated VLDL and IDL cholesterol). Blood samples are taken at multipletime intervals for assessment of radioactivity in plasma.

8.3 STABILITY IN HUMAN SERUM

The stability of the ApoA-I agonists of the invention in human serum isdemonstrated as described below.

8.3.1 EXPERIMENTAL METHODS

100 mg of ¹⁴C-labeled peptide (prepared as described in Section 9.1,supra), is mixed with 2 ml of fresh human plasma (at 37° C.) anddelipidated either immediately (control sample) or after 8 days ofincubation at 37° C. (test sample). Delipidation is carried out byextracting the lipids with an equal volume of 2:1 (v/v)chloroform:methanol.

The samples are loaded onto a reverse-phase C18 HPLC column and elutedwith a linear gradient (25-58% over 33 min.) of acetonitrile (containing0.1% TFA). Elution profiles are followed by absorbance (220 nm) andradioactivity.

8.4 FORMATION OF PRE-β LIKE PARTICLES

The ability of the ApoA-I agonists of the invention to form pre-β-likeparticles is demonstrated as described below.

6.4.1 EXPERIMENTAL METHOD

Human HDL is isolated by KBr density ultra centrifugation at densityd=1.21 g/ml to obtain top fraction followed by Superose 6 gel filtrationchromatography to separate HDL from other lipoproteins. Isolated HDL isadjusted to a final concentration of 1.0 mg/ml with physiological salinebased on protein content determined by Bradford protein assay. Analiquot of 300 μl is removed from the isolated HDL preparation andincubated with 100 μl ¹⁴C-labeled peptide for two hours at 37° C. Fiveseparate incubations are analyzed including a blank containing 100 μlphysiological saline and four dilutions of ¹⁴C-labeled peptide: (i) 0.20μg/μl peptide:HDL, ratio=1:15; (ii) 0.30 μg/μl peptide:HDL, ratio=1:10;(iii) 0.60 μg/μl peptide:HDL, ratio=1:5; and (iv) 1.00 μg/μlpeptide:HDL, ratio=1:3. Following the two hour incubation, a 200 μlaliquot of the sample (total volume=400 μl) is loaded onto a Superose 6gel filtration column for lipoprotein separation and analysis, and 100μl is used to determine total radioactivity loaded onto the column.

8.5 ASSOCIATION OF APo-A-I AGONISTS WITH HUMAN LIPOPROTEINS 8.5.1Experimental Methods

The ability of the ApoA-I agonists of the invention to associate withhuman lipoprotein fractions is determined by incubating ¹⁴C-labeledpeptide with each lipoprotein class (HDL, LDL and VLDL) and a mixture ofthe different lipoprotein classes.

HDL, LDL and VLDL are isolated by KBr density gradientultracentrifugation at d=1.21 g/ml and purified by FPLC on a Superose 6Bcolumn size exclusion column (chromatography is carried out at a flowrate of 0.7 ml/min. and a running buffer of 10 mM Tris (pH 8), 115 mMNaCl, 2 mM EDTA and 0.01% NaN₃). ¹⁴C-labeled peptide is incubated withHDL, LDL and VLDL at a peptide:phospholipid ratio of 1:5 (mass ratio)for 2 h at 37° C. The required amount of lipoprotein (volumes based onamount needed to yield 1000 μg) is mixed with 0.2 ml of peptide stocksolution (1 mg/ml) and the solution is brought up to 2.2 ml using 0.9%of NaCl.

After incubating for 2 hr. at 37° C., an aliquot (0.1 ml) is removed forliquid scintillation counting to determine the total radioactivity, thedensity of the remaining incubation mixture is adjusted to 1.21 g/mlwith KBr, and the samples are centrifuged at 100,000 rpm (300,000 g) for24 hours at 4° C. in a TLA 100.3 rotor using a Beckman tabletopultracentrifuge. The resulting supernatant is fractionated by removing0.3 ml aliquots from the top of each sample for a total of 5 fractions,and 0.05 ml of each fraction is used for liquid scintillation counting.The top two fractions contain the floating lipoproteins, the otherfractions (3-5) correspond to proteins/peptides in solution.

8.6 THE ApoA-I AGONISTS OF THE INVENTION SELECTIVELY BIND HDL LIPIDS INHUMAN PLASMA 8.6.1 Experimental Method

To demonstrate that the ApoA-I agonists of the invention selectivelybind HDL proteins in human plasma, 2 ml of human plasma is incubatedwith 20, 40, 60, 80, and 100 μg of ¹⁴C-labeled peptide for 2 hr. at 37°C. The lipoproteins are separated by adjusting the density to 1.21 g/mland centrifugation in a TLA 100.3 rotor at 100,000 rpm. (300,000 g) for36 hr. at 4° C. The top 900 μl (in 300 μl fractions) is taken foranalysis. 50 μl from each 300 μl fraction is counted for radioactivityand 200 μl from each fraction is analyzed by FPLC (Superose 6/Superose12 combination column).

9 EXAMPLE: THE ApoA-I AGONISTS PROMOTE CHOLESTEROL EFFLUX

To demonstrate that the ApoA-I agonists of the invention promotecholesterol efflux, HepG hepatoma cells are plated into 6-well culturedishes and grown to confluence. Cells are labeled with ³H-cholesterol bydrying the cholesterol, then adding 1% bovine serum albumin (BSA) inphosphate buffered saline (PBS), sonicating the solution, and adding 0.2ml of this labeling solution and 1.8 ml growth medium to the cells, sothat each well contains 2 μCi of radioactivity. Cells are incubated for24 hr. with the labeling medium.

Peptide (or protein):DMPC complexes are prepared at a 1:2 peptide (orprotein):DMPC ratio (w:w). To prepare the complexes, peptide or nativehuman ApoA-I protein is added to a DMPC solution in PBS and incubated atroom temperature overnight, by which time the solution will clarify.Peptide or protein concentration in the final solution is about 1 mg/ml.

Labeling media is removed from the cells and the cells are washed withPBS prior to addition of complexes. 1.6 ml of growth medium is added toeach well, followed by peptide (or protein):DMPC complex and sufficientPBS to bring the final volume to 2 ml per well. The final peptide orApoA-I concentrations are about 1, 2.5, 5, 7.5 and 25 μg/ml medium.After 24 hours of incubation at 37° C., the medium is removed, and thecells are washed with 2 ml of 1% BSA/PBS, followed by 2 washes with 2 mleach of PBS. The amount of ³H-cholesterol effluxed into the medium isdetermined by liquid scintillation counting.

EXAMPLE: USE OF THE ApoA-I AGONISTS IN ANIMAL MODEL SYSTEMS

The efficacy of the ApoA-I agonists of the invention was demonstrated inrabbits. The results show that administration of the ApoA-I agonistsincreases serum concentration of HDL-like particles.

10.1 Preparation of the Phospholipid/Peptide Complexes

Small discoidal particles consisting of phospholipid (DPPC) and peptide146 (SEQ ID NO:146) were prepared following the cholate dialysis method.The phospholipid was dissolved in chloroform and dried under a stream ofnitrogen. The peptide was dissolved in buffer (saline) at aconcentration of 1-2 mg/ml. The lipid film was redissolved in buffercontaining cholate (43° C.) and the peptide solution was added at a 3-1phospholipid/peptide ratio. The mixture was incubated overnight at 43°C. and then dialyzed at 43° C. (24 hr.), room temperature (24 hr.) and4° C. (24 hr.), with three changes of buffer (large volumes) attemperature point. The complexes were filter sterilized (0.22μ) forinjection and storage at 4° C.

10.2 ISOLATION AND CHARACTERIZATION OF THE PEPTIDE/PHOSPHOLIPIDPARTICLES

The particles were separated on a gel filtration column (Superose 6 HR).The position of the peak containing the particles was identified bymeasuring the phospholipid concentration in each fraction. From theelution volume, the Stokes radius can was determined. The concentrationof peptide in the complex was determined by determining thephenylalanine content (by HPLC) following a 16 hr. acid hydrolysis.

10.3 INJECTION IN THE RABBIT

Male New Zealand White rabbits (2.5-3 kg) were injected intravenouslywith a dose of phospholipid/peptide complex (8 mg/kg bodyweight peptide146 (SEQ ID NO:146) or 10 mg/kg bodyweight ApoA-I, expressed as peptideor protein content) in a single bolus injection not exceeding 10-15 ml.The animals were slightly sedated before the manipulations. Bloodsamples (collected on EDTA) were taken before and 5, 15, 30, 60, 240 and1440 minutes after injection. The hematocrit (Hct) was determined foreach sample. Samples were aliquoted and stored at −20° C. beforeanalysis.

10.4 ANALYSIS OF THE RABBIT SERA

Plasma Lipids. The total plasma cholesterol, plasma triglycerides andplasma phospholipids were determined enzymatically using commerciallyavailable assays according to the manufacturer's protocols (BoehringerMannheim, Mannheim, Germany and Biomerieux, 69280, Marcy-l'étoile,France).

Lipoprotein Profiles. The plasma lipoprotein profiles of the fractionsobtained after the separation of the plasma into its lipoproteinfractions were determined by spinning in a sucrose density gradient. Thefractions were collected and in each individual fraction thephospholipid and cholesterol content was measured enzymatically.

10.5 RESULTS

The lipoprotein profile of rabbits injected with 8 mg/kg peptide 146(SEQ ID NO:146) (in the form of peptide/DPPC complexes) as a function oftime. A substantial increase in cholesterol of the HDL cholesterolfractions (fractions >1.06 mg/ml) is apparent at 5 min. followinginjection and lasts for approximately 24 hr.

The cholesterol of the combined HDL fractions obtained by densitygradient ultracentrifugation is presented in Table X, below. The highestincrease of HDL cholesterol (90%) occurred 240 min. afteradministration. At 24 hr. following administration, the increase wasstill 71.2%.

These data indicate that administration of peptide 146/DPPC complexes (8mg/kg) induces rapid and efficient mobilization of peripheralcholesterol. TABLE X HDL CHOLESTEROL IN RABBITS FOLLOWING ADMINISTRATIONOF 8 mg/kg 146 (SEQ ID NO: 146) or 10 mg/kg NATIVE ApoA-I Increase inHDL Increase in HDL Cholesterol (%) Cholesterol (%) Time (min.) NativeApoA-I Peptide 146 5 19.3 31.3 15 16 60.4 60 15.8 42.9 240 −24.1 90.21440 * 71.2* animal died prior to time point

11 EXAMPLE: PREPARATION OF PEPTIDE-LIPID COMPLEX BY CO-LYOPHILIZATIONAPPROACH

The following protocol was utilized to prepare peptide-lipid complexes.

One mg of peptide 149 (PVLELFENLWERLLDALQKKLK; SEQ ID NO:149) wasdissolved in 250 μl HPLC grade methanol (Perkin Elmer) in a one ml clearglass vial with cap (Waters #WAT025054). Dissolving of the peptide wasaided by occasional vortexing over a period of 10 minutes at roomtemperature. To this mixture an aliquot containing 3 mgdipalmitoyl-phosphatidyl-choline (DPPC; Avanti Polar Lipids, 99% Purity,product #850355) from a 100 mg/ml stock solution in methanol was added.The volume of the mixture was brought to 400 μl by addition of methanol,and the mixture was further vortexed intermittently for a period of 10minutes at room temperature. To the tube 200 μl of xylene (Sigma-Aldrich99% pure, HPLC-grade) was added and the tubes were vortexed for 10seconds. Two small holes were punched into the top of the tube with a 20gauge syringe needle, the tube was frozen for 15 seconds in liquidnitrogen, and the tube was lyophilized overnight under vacuum. To thetube 200 mls. of 0.9% NaCl solution was added. The tube was vortexed for20 seconds. At this time the solution in the tube was milky inappearance. The tube was then incubated in a water bath for 30 minutesat 41° C. The solution became clear (i.e., similar to water inappearance) after a few minutes of incubation at 41° C.

11.1 CHARACTERIZATION OF COMPLEXES BY SUPEROSE 6 GEL FILTRATIONCHROMATOGRAPHY

Peptide-phospholipid complexes containing peptide 149 (SEQ ID NO:149)were prepared by colyophilization as described above. The preparationcontained 1 mg peptide and 3 mgs DPPC by weight. After reconstitutingthe complexes in 200 μl of 0.9% NaCl, 20l (containing 100 μg peptide149) of the complexes were applied to a Pharmacia Superose 6 columnusing 0.9% NaCl as the liquid phase at a flow rate of 0.5 mls/minute.The chromatography was monitored by absorbance or scattering of light ofwavelength 280 nm. One ml fractions were collected. Aliquots containing20 μl of the fractions were assayed for phospholipid content using thebioMerieux Phospholipides Enzymatique PAP 150 kit (#61491) according tothe instructions supplied by the manufacturer. The vast majority of bothphospholipid and UV absorbance were recovered together in a fewfractions with peaks at approximately 15.8 mls. This elution volumecorresponds to a Stokes' diameter of 87 Angstroms.

For comparison, a separate chromatogram of 20 μl of human HDL₂ was rununder the same conditions and using the same column as the peptide 149complexes. The HDL₂ was prepared as follows: 300 mls frozen human plasma(Mannheim Blutspendzentrale #1185190) was thawed, adjusted to density1.25 with solid potassium bromide, and centrifuged 45 hours at 40,000RPM using a Ti45. rotor (Beckman) at 20° C. The floating layer wascollected, dialyzed against distilled water, adjusted to density 1.07with solid potassium bromide, and centrifuged as described above for 70hours. The bottom layer (at a level of one cm above the tube bottom) wascollected, brought to 0.01% sodium azide, and stored at 4° C. for 4 daysuntil chromatography. The column eluate was monitored by absorbance orscattering of light of wavelength 254 nm. A series of proteins of knownmolecular weight and Stokes' diameter were used as standards tocalibrate the column for the calculation of Stokes' diameters of theparticles (Pharmacia Gel Filtration Calibration Kit Instruction Manual,Pharmacia Laboratory Separation, Piscataway, N.J., revised April, 1985).The HDL₂ eluted with a retention volume of 14.8 mls, corresponding to aStokes' diameter of 108 nm.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. A nucleotide sequence encoding an An Apo-A-I agonist comprising: (i)a 15 to 29-residue peptide or peptide analogue which forms anamphipathic α-helix in the presence of lipids and which comprises thestructural formula (I):X₁-X₂-X₃-X₄-X₅-X₆-X₇-X8-X₉-XIO-XII-XI₂-XI₃-XI₄-XI₅-X_(I6)-X₁₇-X₁₈-X-₁₉-X₂₀-X₂₁-X₂₂-X₂₃ or apharmaceutically acceptable salt thereof, wherein: X₁ is Ala (A), Gly(G), Gln (Q), Asn (N), Asp (D) or Pro (P); X₂ is an aliphatic residue;X₃ is Leu (L) or Phe (F); X₄ is Glu (E); X₅ is an aliphatic residue; X₆is Leu (L) or Phe (F); X₇ is Glu (E) or Leu (L); X₈ is Asn (N) or Gln(Q); X₉ is Leu (L); X₁₀ is Leu (L), Trp (W) or Gly (G); X₁₁ is an acidicresidue; X₁₂ is Arg (R); X₁₃ is Leu (L) or Gly (G); X₁₄ is Leu (L), Phe(F) or Gly (G); X₁₅ is Asp (D); X₁₆ is Ala (A); X₁₇ is Leu (L); X₁₈ isAsn (N) or Gln (Q); X₁₉ is a basic residue; X₂₀ is a basic residue; X₂₁is Leu (L); X₂₂ is a basic residue; X₂₃ is absent or a basic residue; or(ii) a deleted form of structural formula (I) in which at least one andup to eight of residues X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁,X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀, X₂₁ or X₂₂ are deleted; or(iii) an altered form of structural formula (I) in which at least one ofresidues X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄,X₅, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀, X₂₁, X₂₂ or X₂₃ is conservativelysubstituted with another residue.
 2. (canceled)
 3. The nucleotidesequence of claim 1 encoding an ApoA-I agonist which is the altered formof structural formula (I). 4-18. (canceled)
 19. A nucleotide sequenceencoding an ApoA-I agonist comprising: (i) a 15 to 29-residue peptide orpeptide analogue which forms an amphipathic a-helix in the presence oflipids and which comprises the structural formula (II):X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈-X₁₉-X₂₀-X₂₁-X₂₂-X₂₃or a pharmaceutically acceptable salt thereof, wherein: X₁ is Pro (P),Ala (A), Gly (G), Gln (Q), Asn (N) or Asp (D); X₂ is an aliphaticresidue; X₃ is Leu (L) or Phe (F); X₄ is Glu (E); X₅ is an aliphaticresidue; X₆ is Leu (L) or Phe (F); X₇ is Glu (E) or Leu (L); X₈ is Asn(N) or Gln (Q); X₉ is Leu (L); X₁₀ is Leu (L), Trp (W) or Gly (G); X₁₁is an acidic residue; X₁₂ is Arg (R); X₁₃ is Leu (L) or Gly (G); X₁₄ isLeu (L), Phe (F) or Gly (G); X₁₅ is Asp (D); X₁₆ is Ala (A); X₁₇ is Leu(L); X₁₈ is Asn (N) or Gln (Q); X₁₉ is a basic residue; X₂₀ is a basicresidue; X₂₁ is Leu (L); X₂₂ is a basic residue; X₂₃ is absent or abasic residue; or (ii) a deleted form of structural formula (II) inwhich at least one and up to seven of residues X₁, X₂, X₃, X₄, X₅, X₆,X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, X₁₈, X₁₉, X₂₀, X₂₁and X₂₂ are deleted wherein said structural formula (II) forms anamphipathic α-helix in the presence of lipids; or (iii) an altered formof structural formula (II) in which at least one of residues X₁, X₂, X₃,X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇, X₁₈,X₁₉, X₂₀, X₂₁, X₂₂ and X₂₃ is conservatively substituted with anotherresidue.
 20. (canceled)
 21. The nucleotide sequence of claim 19 (iii)encoding an ApoA-I agonist which is the altered form of structuralformula (II). 22-35. (canceled)
 36. A nucleotide sequence encoding anApoA-I agonist comprising: (i) a 14 to 22-residue peptide or peptideanalogue which forms an amphipathic α-helix in the presence of lipidsand which comprises the structural formula (III):X₁-X₂-X₃-X₄-X₅-X₆-X₇-X₈-X₉-X₁₀-X₁₁-X₁₂-X₁₃-X₁₄-X₁₅-X₁₆-X₁₇-X₁₈ X₁ is Pro(P), Ala (A), Gly (G), Asn (N) or Gln (Q); X₂ is an aliphatic aminoacid; X₃ is Leu (L); X₄ is an acidic amino acid; X₅ is Leu (L) or Phe(F); X₆ is Leu (L) or Phe (F); X₇ is a basic amino acid; X₈ is an acidicamino acid; X₉ is Leu (L) or Trp (W); X₁₀ is Leu (L) or Trp (W): X₁₁ isan acidic amino acid or Asn (N); X₁₂ is an acidic amino acid; X₁₃ is Leu(L), Trp (W) or Phe (F); X₁₄ is a basic amino acid or Leu (L); X₁₅ isGln (Q) or Asn (N); X₁₆ is a basic amino acid; X₁₇ is Leu (L); X₁₈ is abasic amino acid; or (ii) a deleted form of structural formula (III) inwhich at least one and up to seven of residues X₁, X₂, X₃, X₄, X₅, X₆,X₇, X₈, X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇ and X₁₈ are deletedwherein said structural formula (III) forms an amphipathic α-helix inthe presence of lipids; or (iii) an altered form of structural formula(III) in which at least one of residues X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈,X₉, X₁₀, X₁₁, X₁₂, X₁₃, X₁₄, X₁₅, X₁₆, X₁₇ or X₁₈ is conservativelysubstituted with another residue.
 37. (canceled)
 38. The nucleotidesequence of claim 36 (iii) encoding an ApoA-I agonist which is thealtered form of structural formula (III). 39-49. (canceled)
 50. Anucleotide sequence encoding a multimeric ApoA-I agonist which exhibitsat least about 38% LCAT activation activity as compared with humanApoA-I and which has the structural formula (IV):HHLL_(m-)HH_(n)LL_(m-)HH  (IV) or a pharmaceutically acceptable saltthereof, wherein: each m is independently an integer from 0 to 1; n isan integer from 0 to 10; each “LL” is independently a linker; and each“HH” is independently a peptide or peptide analogue according to claim1, 19 or
 36. 51-53. (canceled)
 54. A pharmaceutical compositioncomprising nucleotide sequence encoding an ApoA-I agonist and apharmaceutically acceptable carrier, excipient or diluent, wherein theApoA-I agonist is encoded by the nucleotide sequence of claim 1, 19, 36or
 50. 55-57. (canceled)
 58. A method of treating a subject sufferingfrom a disorder associated with dyslipidemia, said method comprising thestep of administrating to the subject an effective amount of anucleotide sequence or a host cell expressing the nucleotide sequence ofclaims 1, 19, 36 or
 50. 59. The method of claim 58 in which the ApoA-Iagonist is in the form of an ApoA-I agonist-lipid complex, said complexcomprising the ApoA-I agonist and a lipid.
 60. The method of claim 58 inwhich the ApoA-I agonist is in the form of a pharmaceutical composition,said composition comprising the ApoA-I agonist and a pharmaceuticallyacceptable carrier, excipient or diluent.
 61. The method of claim 58 inwhich the disorder associated with dyslipidemia is hypercholesterolemia.62-67. (canceled)
 68. A method of treating a subject suffering from adisorder associated with endotoxemia/septic shock, said methodcomprising the step of administering to the subject an effective amountof a nucleotide sequence or a host cell expressing the nucleotidesequence of claims 1, 19, 36 or
 50. 69. The method of claim 68 in whichthe ApoA-I agonist is in the form of an ApoA-I agonist-lipid complex,said complex comprising the ApoA-I agonist and a lipid.
 70. The methodof claim 68 in which the ApoA-I agonist is in the form of apharmaceutical composition, said composition comprising the ApoA-Iagonist and a pharmaceutically acceptable carrier, excipient or diluent.